@article{ZhangRammingHeinkeetal.2019, author = {Zhang, Yunming and Ramming, Anna and Heinke, Lisa and Altschmied, Lothar and Slotkin, R. Keith and Becker, J{\"o}rg D. and Kappel, Christian and Lenhard, Michael}, title = {The poly(A) polymerase PAPS1 interacts with the RNA-directed DNA-methylation pathway in sporophyte and pollen development}, series = {The plant journal}, volume = {99}, journal = {The plant journal}, number = {4}, publisher = {Wiley}, address = {Hoboken}, issn = {0960-7412}, doi = {10.1111/tpj.14348}, pages = {655 -- 672}, year = {2019}, abstract = {RNA-based processes play key roles in the regulation of eukaryotic gene expression. This includes both the processing of pre-mRNAs into mature mRNAs ready for translation and RNA-based silencing processes, such as RNA-directed DNA methylation (RdDM). Polyadenylation of pre-mRNAs is one important step in their processing and is carried out by three functionally specialized canonical nuclear poly(A) polymerases in Arabidopsis thaliana. Null mutations in one of these, termed PAPS1, result in a male gametophytic defect. Using a fluorescence-labelling strategy, we have characterized this defect in more detail using RNA and small-RNA sequencing. In addition to global defects in the expression of pollen-differentiation genes, paps1 null-mutant pollen shows a strong overaccumulation of transposable element (TE) transcripts, yet a depletion of 21- and particularly 24-nucleotide-long short interfering RNAs (siRNAs) and microRNAs (miRNAs) targeting the corresponding TEs. Double-mutant analyses support a specific functional interaction between PAPS1 and components of the RdDM pathway, as evident from strong synergistic phenotypes in mutant combinations involving paps1, but not paps2 paps4, mutations. In particular, the double-mutant of paps1 and rna-dependent rna polymerase 6 (rdr6) shows a synergistic developmental phenotype disrupting the formation of the transmitting tract in the female gynoecium. Thus, our findings in A. thaliana uncover a potentially general link between canonical poly(A) polymerases as components of mRNA processing and RdDM, reflecting an analogous interaction in fission yeast.}, language = {en} } @article{ThirumalaikumarGorkaSchulzetal.2020, author = {Thirumalaikumar, Venkatesh P. and Gorka, Michal and Schulz, Karina and Masclaux-Daubresse, Celine and Sampathkumar, Arun and Skirycz, Aleksandra and Vierstra, Richard D. and Balazadeh, Salma}, title = {Selective autophagy regulates heat stress memory in Arabidopsis by NBR1-mediated targeting of HSP90.1 and ROF1}, series = {Autophagy}, volume = {17}, journal = {Autophagy}, number = {9}, publisher = {Taylor \& Francis}, address = {Abingdon}, issn = {1554-8635}, doi = {10.1080/15548627.2020.1820778}, pages = {2184 -- 2199}, year = {2020}, abstract = {In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles, plants require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role for macroautophagy/autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS inArabidopsis thaliana. Here, we demonstrated that NBR1 (next to BRCA1 gene 1) plays a crucial role as a receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of the NBR1 protein, NBR1-labeled puncta, and NBR1 activity are all higher during the HS recovery phase than before. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of annbr1-null mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 interacts with HSP90.1 (heat shock protein 90.1) and ROF1 (rotamase FKBP 1), a member of the FKBP family, and mediates their degradation by autophagy, which represses the response to HS by attenuating the expression ofHSPgenes regulated by the HSFA2 transcription factor. Accordingly, loss-of-function mutation ofNBR1resulted in a stronger HS memory phenotype. Together, our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS.}, language = {en} } @article{KuekenGennermannNikoloski2020, author = {K{\"u}ken, Anika and Gennermann, Kristin and Nikoloski, Zoran}, title = {Characterization of maximal enzyme catalytic rates in central metabolism of Arabidopsis thaliana}, series = {The plant journal}, volume = {103}, journal = {The plant journal}, number = {6}, publisher = {Wiley}, address = {Oxford}, issn = {0960-7412}, doi = {10.1111/tpj.14890}, pages = {2168 -- 2177}, year = {2020}, abstract = {Availability of plant-specific enzyme kinetic data is scarce, limiting the predictive power of metabolic models and precluding identification of genetic factors of enzyme properties. Enzyme kinetic data are measuredin vitro, often under non-physiological conditions, and conclusions elicited from modeling warrant caution. Here we estimate maximalin vivocatalytic rates for 168 plant enzymes, including photosystems I and II, cytochrome-b6f complex, ATP-citrate synthase, sucrose-phosphate synthase as well as enzymes from amino acid synthesis with previously undocumented enzyme kinetic data in BRENDA. The estimations are obtained by integrating condition-specific quantitative proteomics data, maximal rates of selected enzymes, growth measurements fromArabidopsis thalianarosette with and fluxes through canonical pathways in a constraint-based model of leaf metabolism. In comparison to findings inEscherichia coli, we demonstrate weaker concordance between the plant-specificin vitroandin vivoenzyme catalytic rates due to a low degree of enzyme saturation. This is supported by the finding that concentrations of nicotinamide adenine dinucleotide (phosphate), adenosine triphosphate and uridine triphosphate, calculated based on our maximalin vivocatalytic rates, and available quantitative metabolomics data are below reportedKMvalues and, therefore, indicate undersaturation of respective enzymes. Our findings show that genome-wide profiling of enzyme kinetic properties is feasible in plants, paving the way for understanding resource allocation.}, language = {en} } @phdthesis{Schaarschmidt2021, author = {Schaarschmidt, Stephanie}, title = {Evaluation and application of omics approaches to characterize molecular responses to abiotic stresses in plants}, doi = {10.25932/publishup-50963}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-509630}, school = {Universit{\"a}t Potsdam}, pages = {viii, 117}, year = {2021}, abstract = {Aufgrund des globalen Klimawandels ist die Gew{\"a}hrleistung der Ern{\"a}hrungssicherheit f{\"u}r eine wachsende Weltbev{\"o}lkerung eine große Herausforderung. Insbesondere abiotische Stressoren wirken sich negativ auf Ernteertr{\"a}ge aus. Um klimaangepasste Nutzpflanzen zu entwickeln, ist ein umfassendes Verst{\"a}ndnis molekularer Ver{\"a}nderungen in der Reaktion auf unterschiedlich starke Umweltbelastungen erforderlich. Hochdurchsatz- oder "Omics"-Technologien k{\"o}nnen dazu beitragen, Schl{\"u}sselregulatoren und Wege abiotischer Stressreaktionen zu identifizieren. Zus{\"a}tzlich zur Gewinnung von Omics-Daten m{\"u}ssen auch Programme und statistische Analysen entwickelt und evaluiert werden, um zuverl{\"a}ssige biologische Ergebnisse zu erhalten. Ich habe diese Problemstellung in drei verschiedenen Studien behandelt und daf{\"u}r zwei Omics-Technologien benutzt. In der ersten Studie wurden Transkript-Daten von den beiden polymorphen Arabidopsis thaliana Akzessionen Col-0 und N14 verwendet, um sieben Programme hinsichtlich ihrer F{\"a}higkeit zur Positionierung und Quantifizierung von Illumina RNA Sequenz-Fragmenten („Reads") zu evaluieren. Zwischen 92\% und 99\% der Reads konnten an die Referenzsequenz positioniert werden und die ermittelten Verteilungen waren hoch korreliert f{\"u}r alle Programme. Bei der Durchf{\"u}hrung einer differentiellen Genexpressionsanalyse zwischen Pflanzen, die bei 20 °C oder 4 °C (K{\"a}lteakklimatisierung) exponiert wurden, ergab sich eine große paarweise {\"U}berlappung zwischen den Programmen. In der zweiten Studie habe ich die Transkriptome von zehn verschiedenen Oryza sativa (Reis) Kultivaren sequenziert. Daf{\"u}r wurde die PacBio Isoform Sequenzierungstechnologie benutzt. Die de novo Referenztranskriptome hatten zwischen 38.900 bis 54.500 hoch qualitative Isoformen pro Sorte. Die Isoformen wurden kollabiert, um die Sequenzredundanz zu verringern und danach evaluiert z.B. hinsichtlich des Vollst{\"a}ndigkeitsgrades (BUSCO), der Transkriptl{\"a}nge und der Anzahl einzigartiger Transkripte pro Genloci. F{\"u}r die hitze- und trockenheitstolerante Sorte N22 wurden ca. 650 einzigartige und neue Transkripte identifiziert, von denen 56 signifikant unterschiedlich in sich entwickelnden Samen unter kombiniertem Trocken- und Hitzestress exprimiert wurden. In der letzten Studie habe ich die Ver{\"a}nderungen in Metabolitprofilen von acht Reissorten gemessen und analysiert, die dem Stress hoher Nachttemperaturen (HNT) ausgesetzt waren und w{\"a}hrend der Trocken- und Regenzeit im Feld auf den Philippinen angebaut wurden. Es wurden jahreszeitlich bedingte Ver{\"a}nderungen im Metabolitspiegel sowie f{\"u}r agronomische Parameter identifiziert und m{\"o}gliche Stoffwechselwege, die einen Ertragsr{\"u}ckgang unter HNT-Bedingungen verursachen, vorgeschlagen. Zusammenfassend konnte ich zeigen, dass der Vergleich der RNA-seq Programme den Pflanzenwissenschaftler*innen helfen kann, sich f{\"u}r das richtige Werkzeug f{\"u}r ihre Daten zu entscheiden. Die de novo Transkriptom-Rekonstruktion von Reissorten ohne Genomsequenz bietet einen gezielten, kosteneffizienten Ansatz zur Identifizierung neuer Gene, die durch verschiedene Stressbedingungen reguliert werden unabh{\"a}ngig vom Organismus. Mit dem Metabolomik-Ansatz f{\"u}r HNT-Stress in Reis habe ich stress- und jahreszeitenspezifische Metabolite identifiziert, die in Zukunft als molekulare Marker f{\"u}r die Verbesserung von Nutzpflanzen verwendet werden k{\"o}nnten.}, language = {en} } @article{MeridaFettke2021, author = {Merida, Angel and Fettke, J{\"o}rg}, title = {Starch granule initiation in Arabidopsis thaliana chloroplasts}, series = {The plant journal}, volume = {107}, journal = {The plant journal}, number = {3}, publisher = {Wiley}, address = {Hoboken}, issn = {0960-7412}, doi = {10.1111/tpj.15359}, pages = {688 -- 697}, year = {2021}, abstract = {The initiation of starch granule formation and the mechanism controlling the number of granules per plastid have been some of the most elusive aspects of starch metabolism. This review covers the advances made in the study of these processes. The analyses presented herein depict a scenario in which starch synthase isoform 4 (SS4) provides the elongating activity necessary for the initiation of starch granule formation. However, this protein does not act alone; other polypeptides are required for the initiation of an appropriate number of starch granules per chloroplast. The functions of this group of polypeptides include providing suitable substrates (maltooligosaccharides) to SS4, the localization of the starch initiation machinery to the thylakoid membranes, and facilitating the correct folding of SS4. The number of starch granules per chloroplast is tightly regulated and depends on the developmental stage of the leaves and their metabolic status. Plastidial phosphorylase (PHS1) and other enzymes play an essential role in this process since they are necessary for the synthesis of the substrates used by the initiation machinery. The mechanism of starch granule formation initiation in Arabidopsis seems to be generalizable to other plants and also to the synthesis of long-term storage starch. The latter, however, shows specific features due to the presence of more isoforms, the absence of constantly recurring starch synthesis and degradation, and the metabolic characteristics of the storage sink organs.}, language = {en} } @article{WangLiMaetal.2021, author = {Wang, Meng and Li, Panpan and Ma, Yao and Nie, Xiang and Grebe, Markus and Men, Shuzhen}, title = {Membrane sterol composition in Arabidopsis thaliana affects root elongation via auxin biosynthesis}, series = {International journal of molecular sciences}, volume = {22}, journal = {International journal of molecular sciences}, number = {1}, publisher = {MDPI}, address = {Basel}, issn = {1422-0067}, doi = {10.3390/ijms22010437}, pages = {20}, year = {2021}, abstract = {Plant membrane sterol composition has been reported to affect growth and gravitropism via polar auxin transport and auxin signaling. However, as to whether sterols influence auxin biosynthesis has received little attention. Here, by using the sterol biosynthesis mutant cyclopropylsterol isomerase1-1 (cpi1-1) and sterol application, we reveal that cycloeucalenol, a CPI1 substrate, and sitosterol, an end-product of sterol biosynthesis, antagonistically affect auxin biosynthesis. The short root phenotype of cpi1-1 was associated with a markedly enhanced auxin response in the root tip. Both were neither suppressed by mutations in polar auxin transport (PAT) proteins nor by treatment with a PAT inhibitor and responded to an auxin signaling inhibitor. However, expression of several auxin biosynthesis genes TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS1 (TAA1) was upregulated in cpi1-1. Functionally, TAA1 mutation reduced the auxin response in cpi1-1 and partially rescued its short root phenotype. In support of this genetic evidence, application of cycloeucalenol upregulated expression of the auxin responsive reporter DR5:GUS (beta-glucuronidase) and of several auxin biosynthesis genes, while sitosterol repressed their expression. Hence, our combined genetic, pharmacological, and sterol application studies reveal a hitherto unexplored sterol-dependent modulation of auxin biosynthesis during Arabidopsis root elongation.}, language = {en} } @article{LiuLiFettke2021, author = {Liu, Qingting and Li, Xiaoping and Fettke, J{\"o}rg}, title = {Starch granules in Arabidopsis thaliana mesophyll and guard cells show similar morphology but differences in size and number}, series = {International journal of molecular sciences}, volume = {22}, journal = {International journal of molecular sciences}, number = {11}, publisher = {Molecular Diversity Preservation International}, address = {Basel}, issn = {1422-0067}, doi = {10.3390/ijms22115666}, pages = {11}, year = {2021}, abstract = {Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.}, language = {en} } @misc{LiuLiFettke2021, author = {Liu, Qingting and Li, Xiaoping and Fettke, J{\"o}rg}, title = {Starch granules in Arabidopsis thaliana mesophyll and guard cells show similar morphology but differences in size and number}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1143}, issn = {1866-8372}, doi = {10.25932/publishup-51106}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-511067}, pages = {13}, year = {2021}, abstract = {Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells.}, language = {en} } @misc{LiuZhouFettke2021, author = {Liu, Qingting and Zhou, Yuan and Fettke, J{\"o}rg}, title = {Starch granule size and morphology of Arabidopsis thaliana starch-related mutants analyzed during diurnal rhythm and development}, series = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, volume = {26}, journal = {Zweitver{\"o}ffentlichungen der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, edition = {19}, publisher = {Universit{\"a}tsverlag Potsdam}, address = {Potsdam}, issn = {1866-8372}, doi = {10.25932/publishup-55029}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-550291}, pages = {1 -- 9}, year = {2021}, abstract = {Transitory starch plays a central role in the life cycle of plants. Many aspects of this important metabolism remain unknown; however, starch granules provide insight into this persistent metabolic process. Therefore, monitoring alterations in starch granules with high temporal resolution provides one significant avenue to improve understanding. Here, a previously established method that combines LCSM and safranin-O staining for in vivo imaging of transitory starch granules in leaves of Arabidopsis thaliana was employed to demonstrate, for the first time, the alterations in starch granule size and morphology that occur both throughout the day and during leaf aging. Several starch-related mutants were included, which revealed differences among the generated granules. In ptst2 and sex1-8, the starch granules in old leaves were much larger than those in young leaves; however, the typical flattened discoid morphology was maintained. In ss4 and dpe2/phs1/ss4, the morphology of starch granules in young leaves was altered, with a more rounded shape observed. With leaf development, the starch granules became spherical exclusively in dpe2/phs1/ss4. Thus, the presented data provide new insights to contribute to the understanding of starch granule morphogenesis.}, language = {en} } @article{LiuZhouFettke2021, author = {Liu, Qingting and Zhou, Yuan and Fettke, J{\"o}rg}, title = {Starch granule size and morphology of Arabidopsis thaliana starch-related mutants analyzed during diurnal rhythm and development}, series = {Molecules : a journal of synthetic chemistry and natural product chemistry / Molecular Diversity Preservation International}, volume = {26}, journal = {Molecules : a journal of synthetic chemistry and natural product chemistry / Molecular Diversity Preservation International}, edition = {19}, publisher = {MDPI}, address = {Basel, Schweiz}, issn = {1420-3049}, doi = {10.3390/molecules26195859}, pages = {1 -- 9}, year = {2021}, abstract = {Transitory starch plays a central role in the life cycle of plants. Many aspects of this important metabolism remain unknown; however, starch granules provide insight into this persistent metabolic process. Therefore, monitoring alterations in starch granules with high temporal resolution provides one significant avenue to improve understanding. Here, a previously established method that combines LCSM and safranin-O staining for in vivo imaging of transitory starch granules in leaves of Arabidopsis thaliana was employed to demonstrate, for the first time, the alterations in starch granule size and morphology that occur both throughout the day and during leaf aging. Several starch-related mutants were included, which revealed differences among the generated granules. In ptst2 and sex1-8, the starch granules in old leaves were much larger than those in young leaves; however, the typical flattened discoid morphology was maintained. In ss4 and dpe2/phs1/ss4, the morphology of starch granules in young leaves was altered, with a more rounded shape observed. With leaf development, the starch granules became spherical exclusively in dpe2/phs1/ss4. Thus, the presented data provide new insights to contribute to the understanding of starch granule morphogenesis.}, language = {en} } @phdthesis{MorenoCurtidor2021, author = {Moreno Curtidor, Catalina}, title = {Elucidating the molecular basis of enhanced growth in the Arabidopsis thaliana accession Bur-0}, doi = {10.25932/publishup-52681}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-526814}, school = {Universit{\"a}t Potsdam}, pages = {136}, year = {2021}, abstract = {The life cycle of flowering plants is a dynamic process that involves successful passing through several developmental phases and tremendous progress has been made to reveal cellular and molecular regulatory mechanisms underlying these phases, morphogenesis, and growth. Although several key regulators of plant growth or developmental phase transitions have been identified in Arabidopsis, little is known about factors that become active during embryogenesis, seed development and also during further postembryonic growth. Much less is known about accession-specific factors that determine plant architecture and organ size. Bur-0 has been reported as a natural Arabidopsis thaliana accession with exceptionally big seeds and a large rosette; its phenotype makes it an interesting candidate to study growth and developmental aspects in plants, however, the molecular basis underlying this big phenotype remains to be elucidated. Thus, the general aim of this PhD project was to investigate and unravel the molecular mechanisms underlying the big phenotype in Bur-0. Several natural Arabidopsis accessions and late flowering mutant lines were analysed in this study, including Bur-0. Phenotypes were characterized by determining rosette size, seed size, flowering time, SAM size and growth in different photoperiods, during embryonic and postembryonic development. Our results demonstrate that Bur-0 stands out as an interesting accession with simultaneously larger rosettes, larger SAM, later flowering phenotype and larger seeds, but also larger embryos. Interestingly, inter-accession crosses (F1) resulted in bigger seeds than the parental self-crossed accessions, particularly when Bur-0 was used as the female parental genotype, suggesting parental effects on seed size that might be maternally controlled. Furthermore, developmental stage-based comparisons revealed that the large embryo size of Bur-0 is achieved during late embryogenesis and the large rosette size is achieved during late postembryonic growth. Interestingly, developmental phase progression analyses revealed that from germination onwards, the length of developmental phases during postembryonic growth is delayed in Bur-0, suggesting that in general, the mechanisms that regulate developmental phase progression are shared across developmental phases. On the other hand, a detailed physiological characterization in different tissues at different developmental stages revealed accession-specific physiological and metabolic traits that underlie accession-specific phenotypes and in particular, more carbon resources during embryonic and postembryonic development were found in Bur-0, suggesting an important role of carbohydrates in determination of the bigger Bur-0 phenotype. Additionally, differences in the cellular organization, nuclei DNA content, as well as ploidy level were analyzed in different tissues/cell types and we found that the large organ size in Bur-0 can be mainly attributed to its larger cells and also to higher cell proliferation in the SAM, but not to a different ploidy level. Furthermore, RNA-seq analysis of embryos at torpedo and mature stage, as well as SAMs at vegetative and floral transition stage from Bur-0 and Col-0 was conducted to identify accession-specific genetic determinants of plant phenotypes, shared across tissues and developmental stages during embryonic and postembryonic growth. Potential candidate genes were identified and further validation of transcriptome data by expression analyses of candidate genes as well as known key regulators of organ size and growth during embryonic and postembryonic development confirmed that the high confidence transcriptome datasets generated in this study are reliable for elucidation of molecular mechanisms regulating plant growth and accession-specific phenotypes in Arabidopsis. Taken together, this PhD project contributes to the plant development research field providing a detailed analysis of mechanisms underlying plant growth and development at different levels of biological organization, focusing on Arabidopsis accessions with remarkable phenotypical differences. For this, the natural accession Bur-0 was an ideal outlier candidate and different mechanisms at organ and tissue level, cell level, metabolism, transcript and gene expression level were identified, providing a better understanding of different factors involved in plant growth regulation and mechanisms underlying different growth patterns in nature.}, language = {en} } @phdthesis{Vyse2022, author = {Vyse, Kora}, title = {Elucidating molecular determinants of the loss of freezing tolerance during deacclimation after cold priming and low temperature memory after triggering}, school = {Universit{\"a}t Potsdam}, pages = {vii, 147}, year = {2022}, abstract = {W{\"a}hrend ihrer Entwicklung m{\"u}ssen sich Pflanzen an Temperaturschwankungen anpassen. Niedrige Temperaturen {\"u}ber dem Gefrierpunkt induzieren in Pflanzen eine K{\"a}lteakklimatisierung und h{\"o}here Frosttoleranz, die sich bei w{\"a}rmeren Temperaturen durch Deakklimatisierung wieder zur{\"u}ckbildet. Der Wechsel zwischen diesen beiden Prozessen ist f{\"u}r Pflanzen unerl{\"a}sslich, um als Reaktion auf unterschiedliche Temperaturbedingungen eine optimale Fitness zu erreichen. Die K{\"a}lteakklimatisierung ist umfassend untersucht worden,{\"u}ber die Regulierung der Deakklimatisierung ist jedoch wenig bekannt. In dieser Arbeit wird der Prozess der Deakklimatisierung auf physiologischer und molekularer Ebene in Arabidopsis thaliana untersucht. Messungen des Elektrolytverlustes w{\"a}hrend der K{\"a}lteakklimatisierung und bis zu vier Tagen nach Deakklimatisierung erm{\"o}glichten die Identifizierung von vier Knockout-Mutanten (hra1, lbd41, mbf1c und jub1), die im Vergleich zum Wildtyp eine langsamere Deakklimatisierungsrate aufwiesen. Eine transkriptomische Studie mit Hilfe von RNA-Sequenzierung von A. thaliana Col-0, jub1 und mbf1c zeigte die Bedeutung der Hemmung von stressreaktiven und Jasmonat-ZIM-Dom{\"a}nen-Genen sowie die Regulierung von Zellwandmodifikationen w{\"a}hrend der Deakklimatisierung. Dar{\"u}ber hinaus zeigten Messungen der Alkoholdehydrogenase Aktivit{\"a}t und der Genexpressions{\"a}nderungen von Hypoxiemarkern w{\"a}hrend der ersten vier Tagen der Deakklimatisierung, dass eine Hypoxie-Reaktion w{\"a}hrend der Deakklimatisierung aktiviert wird. Es wurde gezeigt, dass die epigenetische Regulierung w{\"a}hrend der K{\"a}lteakklimatisierung und der 24-st{\"u}ndigen Deakklimatisierung in A. thaliana eine große Rolle spielt. Dar{\"u}ber hinaus zeigten beide Deakklimatisierungsstudien, dass die fr{\"u}here Hypothese, dass Hitzestress eine Rolle bei der fr{\"u}hen Deakklimatisierung spielen k{\"o}nnte, unwahrscheinlich ist. Eine Reihe von DNA- und Histondemethylasen sowie Histonvarianten wurden w{\"a}hrend der Deakklimatisierung hochreguliert, was auf eine Rolle im pflanzlichen Ged{\"a}chtnis schließen l{\"a}sst. In j{\"u}ngster Zeit haben mehrere Studien gezeigt, dass Pflanzen in der Lage sind, die Erinnerung an einen vorangegangenen K{\"a}ltestress auch nach einer Woche Deakklimatisierung zu bewahren. In dieser Arbeit ergaben Transkriptom- und Metabolomanalysen von Arabidopsis w{\"a}hrend 24 Stunden Priming (K{\"a}lteakklimatisierung) und Triggering (wiederkehrender K{\"a}ltestress nach Deakklimatisierung) eine unikale signifikante und vor{\"u}bergehende Induktion der Transkriptionsfaktoren DREB1D, DREB1E und DREB1F w{\"a}hrend des Triggerings, die zur Feinabstimmung der zweiten K{\"a}ltestressreaktion beitr{\"a}gt. Dar{\"u}ber hinaus wurden Gene, die f{\"u}r Late Embryogenesis Abundant (LEA) und Frostschutzproteine kodieren, sowie Proteine, die reaktive Sauerstoffspezies entgiften, w{\"a}hrend des sp{\"a}ten Triggerings (24 Stunden) st{\"a}rker induziert als nach dem ersten K{\"a}lteimpuls, w{\"a}hrend Xyloglucan- Endotransglucosylase/Hydrolase Gene, deren Produkte f{\"u}r eine Restrukturierung der Zellwand verantwortlich sind, fr{\"u}h auf das Triggering reagierten. Die starke Induktion dieser Gene, sowohl bei der Deakklimatisierung als auch beim Triggering, l{\"a}sst vermuten, dass sie eine wesentliche Rolle bei der Stabilisierung der Zellen w{\"a}hrend des Wachstums und bei der Reaktion auf wiederkehrende Stressbedingungen spielen. Zusammenfassend gibt diese Arbeit neue Einblicke in die Regulierung der Deakklimatisierung und des K{\"a}ltestress-Ged{\"a}chtnisses in A. thaliana und er{\"o}ffnet neue M{\"o}glichkeiten f{\"u}r k{\"u}nftige, gezielte Studien von essentiellen Genen in diesem Prozess.}, language = {en} } @article{RalevskiApeltOlasetal.2022, author = {Ralevski, Alexandra and Apelt, Federico and Olas, Justyna Jadwiga and M{\"u}ller-R{\"o}ber, Bernd and Rugarli, Elena I. and Kragler, Friedrich and Horvath, Tamas L.}, title = {Plant mitochondrial FMT and its mammalian homolog CLUH controls development and behavior in Arabidopsis and locomotion in mice}, series = {Cellular and molecular life sciences}, volume = {79}, journal = {Cellular and molecular life sciences}, number = {6}, publisher = {Springer International Publishing AG}, address = {Cham (ZG)}, issn = {1420-682X}, doi = {10.1007/s00018-022-04382-3}, pages = {17}, year = {2022}, abstract = {Mitochondria in animals are associated with development, as well as physiological and pathological behaviors. Several conserved mitochondrial genes exist between plants and higher eukaryotes. Yet, the similarities in mitochondrial function between plant and animal species is poorly understood. Here, we show that FMT (FRIENDLY MITOCHONDRIA) from Arabidopsis thaliana, a highly conserved homolog of the mammalian CLUH (CLUSTERED MITOCHONDRIA) gene family encoding mitochondrial proteins associated with developmental alterations and adult physiological and pathological behaviors, affects whole plant morphology and development under both stressed and normal growth conditions. FMT was found to regulate mitochondrial morphology and dynamics, germination, and flowering time. It also affects leaf expansion growth, salt stress responses and hyponastic behavior, including changes in speed of hyponastic movements. Strikingly, Cluh(+/-) heterozygous knockout mice also displayed altered locomotive movements, traveling for shorter distances and had slower average and maximum speeds in the open field test. These observations indicate that homologous mitochondrial genes may play similar roles and affect homologous functions in both plants and animals.}, language = {en} } @phdthesis{Mahto2022, author = {Mahto, Harendra}, title = {In vitro analysis of Early Starvation 1 (ESV1) and Like Early Starvation 1 (LESV) on starch degradation with focus on glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD)}, pages = {167}, year = {2022}, abstract = {Starch is an insoluble polyglucan, comprises of two polymers, namely, the branched α-1,4: α-1,6-D-glucan amylopectin and the almost unbranched α-1,4-D-glucan amylose. The growth of all plants is directly dependent on the accumulation of transitory starch during the daytime when photosynthesis takes place and subsequently starch degradation during the night. Starch phosphorylation takes place by starch-related dikinases called α-glucan, water dikinase (GWD), and phosphoglucan, water dikinase (PWD), and is a very important step in starch degradation. The biochemical mechanisms of phosphorylation of starch are not properly understood. Recent studies have found that there are two starch binding proteins namely, Early Starvation1 (ESV1) and Like Early Starvation1 (LESV), which play an important role in starch metabolism. It has been shown that ESV1 and LESV proteins affect the starch phosphorylation activity of GWD and PWD enzymes, which control the rate of degradation of starch granules. In this thesis, various in vitro assays were performed to identify and understand the mechanism of recombinant proteins; ESV1 and LESV on the starch degradation. The starch degradation was performed by phosphorylation enzymes, GWD and PWD separately. In various enzymatic assays, the influence of the ESV1 and LESV on the actions of GWD and PWD on the surfaces of different native starch granules were analysed. Furthermore, ESV1 and LESV have specifically shown influences on the phosphorylation activities of GWD and PWD on the starch granule surfaces in an antagonistic pattern in such a way that, the GWD mediated phosphorylation were significantly reduced while PWD mediated phosphorylation were significantly increased respectively. In another set of experiments, ISA and BAM hydrolyzing enzymes were used to alter the structure of starch, and then determine the effect of both dikinases mediated phosphorylation in the presence of ESV1 and LESV on the altered starch granules surfaces. In these results, significant decreases in both GWD and PWD mediated phosphorylation were observed in all the treatments containing either ESV1 or LESV proteins only or both ESV1 and LESV. It was also found that LESV preferentially binds to both amylose and amylopectin, while ESV1 binds to highly ordered glucans such as maltodextrins and amylopectin, which are crystalline in structure. Both ESV1 or LESV proteins either individually or in combination have shown influence on the activity of GWD and PWD phosphate incorporation into the starch granules via reduction even though at different percentages depending on the sources of starch, therefore it is difficult to distinguish the specific function between them. The biochemical studies have shown that protein-glucan interaction specifically between ESV1 or LESV or in combination with different species of starch granules has very strong surface binding, or it might be possible that both the proteins not only bind to the surface of the starch granules but also have entered deep inside the glucan structure of the starch granules. However, the results also revealed that ESV1 and LESV did not alter the autophosphorylation of the dikinases. Also, the chain length distribution pattern of the released glucan chains after treatment of starch with ISA enzyme was evaluated with respect to the degree of polymerization (DP) of the different starch granules. Capillary electrophoresis was employed to study the effect of LESV and ESV1 on the chain length distribution. In summary, this study confirms that ESV1 and LESV play an important role in organizing and regulating the starch metabolism process. In the later half, studies were performed to monitor whether the metabolism of carbohydrates and partitioning, contribute to the higher salt tolerance of the facultative halophyte Hordeum marinum when compared to glycophyte Hordeum vulgare. Seedlings with the same size from both species were hydroponically grown at 0, 150, and 300 mM of NaCl for 3 weeks. H. marinum maintained a high relative growth rate, which was found concomitant in higher aptitude plants to maintain efficient shoot tissue hydration and integrity of membrane under salt conditions when compared to H. vulgare. Hence, our data suggested that the change in the starch storage, distribution of soluble sugar concentrations between source and sink organs, and also changes in the level of enzymes involved in the starch metabolism was significant to give insights into the importance of carbohydrate metabolism in barley species with regards to the salt tolerance. Although these results are still in their nascent state, it could be vital for other researchers to formulate future studies. The preliminary results which were studies about the carbohydrate metabolism and partitioning in salt responses in the halophyte H. marinum and the glycophyte H. vulgare revealed that salt tolerance in barley species is not due to osmotic adjustments, but due to other reasons that were not explored in the past studies. However, the activity of DPE2 in H. vulgare was not hampered by the presence of NaCl as observed. While Pho1 and Pho2, activities were highly increased in cultivated barley. These findings could be suggestive of a possible role of these enzymes in the responses of carbohydrate metabolism to salinity. When sea and cultivated barley species were compared, it was discovered that the former had more versatility in carbohydrate metabolism and distribution.}, language = {en} } @phdthesis{Oberkofler2022, author = {Oberkofler, Vicky}, title = {Molecular basis of HS memory in Arabidopsis thaliana}, doi = {10.25932/publishup-56954}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-569544}, school = {Universit{\"a}t Potsdam}, pages = {181}, year = {2022}, abstract = {Plants can be primed to survive the exposure to a severe heat stress (HS) by prior exposure to a mild HS. The information about the priming stimulus is maintained by the plant for several days. This maintenance of acquired thermotolerance, or HS memory, is genetically separable from the acquisition of thermotolerance itself and several specific regulatory factors have been identified in recent years. On the molecular level, HS memory correlates with two types of transcriptional memory, type I and type II, that characterize a partially overlapping subset of HS-inducible genes. Type I transcriptional memory or sustained induction refers to the sustained transcriptional induction above non-stressed expression levels of a gene for a prolonged time period after the end of the stress exposure. Type II transcriptional memory refers to an altered transcriptional response of a gene after repeated exposure to a stress of similar duration and intensity. In particular, enhanced re-induction refers to a transcriptional pattern in which a gene is induced to a significantly higher degree after the second stress exposure than after the first. This thesis describes the functional characterization of a novel positive transcriptional regulator of type I transcriptional memory, the heat shock transcription factor HSFA3, and compares it to HSFA2, a known positive regulator of type I and type II transcriptional memory. It investigates type I transcriptional memory and its dependence on HSFA2 and HSFA3 for the first time on a genome-wide level, and gives insight on the formation of heteromeric HSF complexes in response to HS. This thesis confirms the tight correlation between transcriptional memory and H3K4 hyper-methylation, reported here in a case study that aimed to reduce H3K4 hyper-methylation of the type II transcriptional memory gene APX2 by CRISPR/dCas9-mediated epigenome editing. Finally, this thesis gives insight into the requirements for a heat shock transcription factor to function as a positive regulator of transcriptional memory, both in terms of its expression profile and protein abundance after HS and the contribution of individual functional domains. In summary, this thesis contributes to a more detailed understanding of the molecular processes underlying transcriptional memory and therefore HS memory, in Arabidopsis thaliana.}, language = {en} } @article{MuntahaLiCompartetal.2022, author = {Muntaha, Sidratul Nur and Li, Xiaoping and Compart, Julia and Apriyanto, Ardha and Fettke, J{\"o}rg}, title = {Carbon pathways during transitory starch degradation in Arabidopsis differentially affect the starch granule number and morphology in the dpe2/phs1 mutant background}, series = {Plant physiology and biochemistry : an official journal of the Federation of European Societies of Plant Physiology}, volume = {180}, journal = {Plant physiology and biochemistry : an official journal of the Federation of European Societies of Plant Physiology}, publisher = {Elsevier}, address = {Paris}, issn = {0981-9428}, doi = {10.1016/j.plaphy.2022.03.033}, pages = {35 -- 41}, year = {2022}, abstract = {The Arabidopsis knockout mutant lacking both the cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) had a dwarf-growth phenotype, a reduced and uneven distribution of starch within the plant rosettes, and a lower starch granule number per chloroplast under standard growth conditions. In contrast, a triple mutant impaired in starch degradation by its additional lack of the glucan, water dikinase (GWD) showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to the wild type. We concluded that ongoing starch degradation is mainly responsible for the observed phenotype of dpe2/phs1. Next, we generated two further triple mutants lacking either the phosphoglucan, water dikinase (PWD), or the disproportionating enzyme 1 (DPE1) in the background of the double mutant. Analysis of the starch metabolism revealed that even minor ongoing starch degradation observed in dpe2/phs1/pwd maintained the double mutant phenotype. In contrast, an additional blockage in the glucose pathway of starch breakdown, as in dpe2/phs1/ dpe1, resulted in a nearly starch-free phenotype and massive chloroplast degradation. The characterized mutants were discussed in the context of starch granule formation.}, language = {en} } @phdthesis{MartinezSeidel2023, author = {Martinez-Seidel, Federico}, title = {Ribosome Heterogeneity and Specialization during Temperature Acclimation in Plants}, doi = {10.25932/publishup-58072}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-580724}, school = {Universit{\"a}t Potsdam}, pages = {374}, year = {2023}, abstract = {Ribosomes decode mRNA to synthesize proteins. Ribosomes, once considered static, executing machines, are now viewed as dynamic modulators of translation. Increasingly detailed analyses of structural ribosome heterogeneity led to a paradigm shift toward ribosome specialization for selective translation. As sessile organisms, plants cannot escape harmful environments and evolved strategies to withstand. Plant cytosolic ribosomes are in some respects more diverse than those of other metazoans. This diversity may contribute to plant stress acclimation. The goal of this thesis was to determine whether plants use ribosome heterogeneity to regulate protein synthesis through specialized translation. I focused on temperature acclimation, specifically on shifts to low temperatures. During cold acclimation, Arabidopsis ceases growth for seven days while establishing the responses required to resume growth. Earlier results indicate that ribosome biogenesis is essential for cold acclimation. REIL mutants (reil-dkos) lacking a 60S maturation factor do not acclimate successfully and do not resume growth. Using these genotypes, I ascribed cold-induced defects of ribosome biogenesis to the assembly of the polypeptide exit tunnel (PET) by performing spatial statistics of rProtein changes mapped onto the plant 80S structure. I discovered that growth cessation and PET remodeling also occurs in barley, suggesting a general cold response in plants. Cold triggered PET remodeling is consistent with the function of Rei-1, a REIL homolog of yeast, which performs PET quality control. Using seminal data of ribosome specialization, I show that yeast remodels the tRNA entry site of ribosomes upon change of carbon sources and demonstrate that spatially constrained remodeling of ribosomes in metazoans may modulate protein synthesis. I argue that regional remodeling may be a form of ribosome specialization and show that heterogeneous cytosolic polysomes accumulate after cold acclimation, leading to shifts in the translational output that differs between wild-type and reil-dkos. I found that heterogeneous complexes consist of newly synthesized and reused proteins. I propose that tailored ribosome complexes enable free 60S subunits to select specific 48S initiation complexes for translation. Cold acclimated ribosomes through ribosome remodeling synthesize a novel proteome consistent with known mechanisms of cold acclimation. The main hypothesis arising from my thesis is that heterogeneous/ specialized ribosomes alter translation preferences, adjust the proteome and thereby activate plant programs for successful cold acclimation.}, language = {en} } @phdthesis{vonBismarck2023, author = {von Bismarck, Thekla}, title = {The influence of long-term light acclimation on photosynthesis in dynamic light}, school = {Universit{\"a}t Potsdam}, pages = {x, 163}, year = {2023}, abstract = {Photosynthesis converts light into metabolic energy which fuels plant growth. In nature, many factors influence light availability for photosynthesis on different time scales, from shading by leaves within seconds up to seasonal changes over months. Variability of light energy supply for photosynthesis can limit a plant´s biomass accumulation. Plants have evolved multiple strategies to cope with strongly fluctuation light (FL). These range from long-term optimization of leaf morphology and physiology and levels of pigments and proteins in a process called light acclimation, to rapid changes in protein activity within seconds. Therefore, uncovering how plants deal with FL on different time scales may provide key ideas for improving crop yield. Photosynthesis is not an isolated process but tightly integrates with metabolism through mutual regulatory interactions. We thus require mechanistic understanding of how long-term light acclimation shapes both, dynamic photosynthesis and its interactions with downstream metabolism. To approach this, we analyzed the influence of growth light on i) the function of known rapid photosynthesis regulators KEA3 and VCCN1 in dynamic photosynthesis (Chapter 2-3) and ii) the interconnection of photosynthesis with photorespiration (PR; Chapter 4). We approached topic (i) by quantifying the effect of different growth light regimes on photosynthesis and photoprotection by using kea3 and vccn1 mutants. Firstly, we found that, besides photosynthetic capacity, the activities of VCCN1 and KEA3 during a sudden high light phase also correlated with growth light intensity. This finding suggests regulation of both proteins by the capacity of downstream metabolism. Secondly, we showed that KEA3 accelerated photoprotective non-photochemical quenching (NPQ) kinetics in two ways: Directly via downregulating the lumen proton concentration and thereby de-activating pH-dependent NPQ, and indirectly via suppressing accumulation of the photoprotective pigment zeaxanthin. For topic (ii), we analyzed the role of PR, a process which recycles a toxic byproduct of the carbon fixation reactions, in metabolic flexibility in a dynamically changing light environment. For this we employed the mutants hpr1 and ggt1 with a partial block in PR. We characterized the function of PR during light acclimation by tracking molecular and physiological changes of the two mutants. Our data, in contrast to previous reports, disprove a generally stronger physiological relevance of PR under dynamic light conditions. Additionally, the two different mutants showed pronounced and distinct metabolic changes during acclimation to a condition inducing higher photosynthetic activity. This underlines that PR cannot be regarded purely as a cyclic detoxification pathway for 2PG. Instead, PR is highly interconnected with plant metabolism, with GGT1 and HPR1 representing distinct metabolic modulators. In summary, the presented work provides further insight into how energetic and metabolic flexibility is ensured by short-term regulators and PR during long-term light acclimation.}, language = {en} } @phdthesis{Apriyanto2023, author = {Apriyanto, Ardha}, title = {Analysis of starch metabolism in source and sink tissue of plants}, school = {Universit{\"a}t Potsdam}, pages = {166}, year = {2023}, abstract = {Starch is an essential biopolymer produced by plants. Starch can be made inside source tissue (such as leaves) and sink tissue (such as fruits and tubers). Nevertheless, understanding how starch metabolism is regulated in source and sink tissues is fundamental for improving crop production. Despite recent advances in the understanding of starch and its metabolism, there is still a knowledge gap in the source and sink metabolism. Therefore, this study aimed to summarize the state of the art regarding starch structure and metabolism inside plants. In addition, this study aimed to elucidate the regulation of starch metabolism in the source tissue using the leaves of a model organism, Arabidopsis thaliana, and the sink tissue of oil palm (Elaeis guineensis) fruit as a commercial crop. The research regarding the source tissue will focus on the effect of the blockage of starch degradation on the starch parameter in leaves, especially in those of A. thaliana, which lack both disproportionating enzyme 2 (DPE2) and plastidial glucan phosphorylase 1 (PHS1) (dpe2/phs1). The additional elimination of phosphoglucan water dikinase (PWD), starch excess 4 (SEX4), isoamylase 3 (ISA3), and disproportionating enzyme 1 (DPE1) in the dpe2/phs1 mutant background demonstrates the alteration of starch granule number per chloroplast. This study provides insights into the control mechanism of granule number regulation in the chloroplast. The research regarding the sink tissue will emphasize the relationship between starch metabolism and the lipid metabolism pathway in oil palm fruits. This study was conducted to observe the alteration of starch parameters, metabolite abundance, and gene expression during oil palm fruit development with different oil yields. This study shows that starch and sucrose can be used as biomarkers for oil yield in oil palms. In addition, it is revealed that the enzyme isoforms related to starch metabolism influence the oil production in oil palm fruit. Overall, this thesis presents novel information regarding starch metabolism in the source tissue of A.thaliana and the sink tissue of E.guineensis. The results shown in this thesis can be applied to many applications, such as modifying the starch parameter in other plants for specific needs.}, language = {en} } @article{KappelFriedrichOberkofleretal.2023, author = {Kappel, Christian and Friedrich, Thomas and Oberkofler, Vicky and Jiang, Li and Crawford, Tim and Lenhard, Michael and B{\"a}urle, Isabel}, title = {Genomic and epigenomic determinants of heat stress-induced transcriptional memory in Arabidopsis}, series = {Genome biology : biology for the post-genomic era}, volume = {24}, journal = {Genome biology : biology for the post-genomic era}, number = {1}, publisher = {BioMed Central}, address = {London}, issn = {1474-760X}, doi = {10.1186/s13059-023-02970-5}, pages = {23}, year = {2023}, abstract = {Background Transcriptional regulation is a key aspect of environmental stress responses. Heat stress induces transcriptional memory, i.e., sustained induction or enhanced re-induction of transcription, that allows plants to respond more efficiently to a recurrent HS. In light of more frequent temperature extremes due to climate change, improving heat tolerance in crop plants is an important breeding goal. However, not all heat stress-inducible genes show transcriptional memory, and it is unclear what distinguishes memory from non-memory genes. To address this issue and understand the genome and epigenome architecture of transcriptional memory after heat stress, we identify the global target genes of two key memory heat shock transcription factors, HSFA2 and HSFA3, using time course ChIP-seq. Results HSFA2 and HSFA3 show near identical binding patterns. In vitro and in vivo binding strength is highly correlated, indicating the importance of DNA sequence elements. In particular, genes with transcriptional memory are strongly enriched for a tripartite heat shock element, and are hallmarked by several features: low expression levels in the absence of heat stress, accessible chromatin environment, and heat stress-induced enrichment of H3K4 trimethylation. These results are confirmed by an orthogonal transcriptomic data set using both de novo clustering and an established definition of memory genes. Conclusions Our findings provide an integrated view of HSF-dependent transcriptional memory and shed light on its sequence and chromatin determinants, enabling the prediction and engineering of genes with transcriptional memory behavior.}, language = {en} }