570 Biowissenschaften; Biologie
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Institute
The importance of cryptic diversity in rotifers is well understood regarding its ecological consequences, but there remains an in depth comprehension of the underlying molecular mechanisms and forces driving speciation. Temperature has been found several times to affect species spatio-temporal distribution and organisms’ performance, but we lack information on the mechanisms that provide thermal tolerance to rotifers. High cryptic diversity was found recently in the freshwater rotifer “Brachionus calyciflorus”, showing that the complex comprises at least four species: B. calyciflorus sensu stricto (s.s.), B. fernandoi, B. dorcas, and B. elevatus. The temporal succession among species which have been observed in sympatry led to the idea that temperature might play a crucial role in species differentiation.
The central aim of this study was to unravel differences in thermal tolerance between species of the former B. calyciflorus species complex by comparing phenotypic and gene expression responses. More specifically, I used the critical maximum temperature as a proxy for inter-species differences in heat-tolerance; this was modeled as a bi-dimensional phenotypic trait taking into consideration the intention and the duration of heat stress. Significant differences on heat-tolerance between species were detected, with B. calyciflorus s.s. being able to tolerate higher temperatures than B. fernandoi.
Based on evidence of within species neutral genetic variation, I further examined adaptive genetic variability within two different mtDNA lineages of the heat tolerant B. calyciflorus s.s. to identify SNPs and genes under selection that might reflect their adaptive history. These analyses did not reveal adaptive genetic variation related to heat, however, they show putatively adaptive genetic variation which may reflect local adaptation. Functional enrichment of putatively positively selected genes revealed signals of adaptation in genes related to “lipid metabolism”, “xenobiotics biodegradation and metabolism” and “sensory system”, comprising candidate genes which can be utilized in studies on local adaptation. An absence of genetically-based differences in thermal adaptation between the two mtDNA lineages, together with our knowledge that B. calyciflorus s.s. can withstand a broad range of temperatures, led to the idea to further investigate shared transcriptomic responses to long-term exposure to high and low temperatures regimes. With this, I identified candidate genes that are involved in the response to temperature imposed stress. Lastly, I used comparative transcriptomics to examine responses to imposed heat-stress in heat-tolerant and heat-sensitive Brachionus species. I found considerably different patterns of gene expression in the two species. Most striking are patterns of expression regarding the heat shock proteins (hsps) between the two species. In the heat-tolerant, B. calyciflorus s.s., significant up-regulation of hsps at low temperatures was indicative of a stress response at the cooler end of the temperature regimes tested here. In contrast, in the heat-sensitive B. fernandoi, hsps generally exhibited up-regulation of these genes along with rising temperatures. Overall, identification of differences in expression of genes suggests suppression of protein biosynthesis to be a mechanism to increase thermal tolerance. Observed patterns in population growth are correlated with the hsp gene expression differences, indicating that this physiological stress response is indeed related to phenotypic life history performance.
ATP-binding cassette (ABC) transporters are present in all kingdoms of life and enable active transport of various different molecules across biological membranes. They all share an overall architecture of two lipophilic transmembrane spanning domains (TMDs) traversing the membrane and two hydrophilic nucleotide binding domains (NBDs) usually lacking sequence identity. The multiplicity in transported molecules is accompanied by extreme diversity in TMDs. Human mitochondria harbor four ABC transporters, namely ABCB6, ABCB7, ABCB8 and ABCB10 with functional homologues in yeast and plants. Except the ones found in Rickettsiae and related bacteria mitochondrial ABC transporters are absent in bacteria. In addition to converting energy mitochondria are important platforms for biosynthesizing various cofactors as iron sulfur clusters, molybdenum cofactor (Moco) or heme. ABCB7 (Atm1 in yeast) has been shown to connect mitochondrial with cytosolic iron sulfur cluster assembly by exporting a yet unknown sulfur containing molecule. In addition, TMDs of Atm1 display a glutathione binding pocket accessible from the matrix which has been identified in all ABCB7-like transporters and also exists in a bacterial ABC transporter homologue of Atm1 in Novosphingobium aromaticivorans. In addition, ATM3, a plant mitochondrial homologous ABC transporter to human ABCB7, has been associated with biosynthesizing Moco.
In this study we used the α-proteobacterium Rhodobacter capsulatus as a model organism to characterize mitochondrial ABC transporter homologues. R. capsulatus contains two homologues to mitochondrial ABC transporters with the corresponding gene loci rcc03139 and rcc02305. They share 38 to 47 % sequence identities to human mitochondrial ABC transporters ABCB8/ABCB10 and ABCB7/ABCB6, respectively. We created interposon mutants lacking either rcc03139 or rcc02305, analyzed the physiological effects on R. capsulatus and compared the findings especially to eukaryotic deletion studies. A viable bacterial double mutant strain lacking both mitochondrial ABC transporters was constructed to investigate possible overlapping functions. Both R. capsulatus single mutants showed a severe accumulation of intracellular reactive oxygen species (ROS) in comparison to ∆nifDK which revealed to be additive in the double mutant. In the proteome of ∆rcc03139I abundancies of tetrapyrrole related proteins were significantly increased in comparison to the proteome of parental strain, which was further validated by reduced amounts of tetrapyrrole intermediates in ∆rcc03139. In contrast, in ∆rcc02305I total glutathione (GSH) was elevated when endogenous GSH biosynthesis was inhibited. In conjunction with proteomic studies we uncovered misbalanced sulfur distribution in ∆rcc02305I. Furthermore, strains lacking Rcc02305 accumulated cyclic pyranopterin monophosphate (cPMP), an intermediate of Moco biosynthesis, as it was already shown for the deletion strain of the eukaryotic counterpart ATM3 in plants. In contrast single mutant strain Δrcc03139I neither accumulated cPMP nor glutathione.
Bioinformatic analysis of the amino acid sequence of Rcc02305 revealed a pyridoxal 5´phosphate (PLP) binding site which overlaps with Walker A within the NBDs of Rcc02305 and other ABCB7-like transporters. The PLP cofactor is well studied in C-DES (L-cysteine/cystine lyase from Synechocystis) for persulfide production and in L-cysteine desulfurases such as IscS and NFS1 for its role in formation of protein-bound persulfides. Based on our findings we are able to propose a new modality for the transport of the sulfur containing molecule: first of all, the transporter produces a highly reactive persulfide which is then subsequently trapped by glutathione polysulfide, already bound within the binding pocket in TMDs. Walker A becomes accessible for ATP and after hydrolysis the mixed polysulfide is released.
Based on our studies we are convinced that both mitochondrial ABC transporter homologues fulfil distinct roles in R. capsulatus: Rcc02305 is a representative of Atm1/ABCB7-like transporters and important for proper sulfur distribution by exporting persulfides. In contrast Rcc03139 is a representative of ABCB6/ABCB10 related transporters and involved in biosynthesizing tetrapyrroles.
Im Mittelpunkt dieser Arbeit standen Analysen zur Charakterisierung der periplasmatischen Aldehyd Oxidoreduktase aus E. coli. Kinetische Untersuchungen mit Ferricyanid als Elektronenakzeptor unter anaeroben Bedingungen zeigten für dieses Enzym eine höhere Aktivität als unter aeroben Bedingungen. Die getroffene Hypothese, dass PaoABC fähig ist Elektronen an molekularen Sauerstoff weiter zu geben, konnte bestätigt werden. Für den Umsatz aromatischer Aldehyde mit molekularem Sauerstoff wurde ein Optimum von pH 6,0 ermittelt. Dies steht im Gegensatz zur Reaktion mit Ferricyanid, mit welchem ein pH-Optimum von 4,0 gezeigt wurde. Die Reaktion von PaoABC mit molekularem Sauerstoff generiert zwar Wasserstoffperoxid, die Produktion von Superoxid konnte dagegen nicht beobachtet werden. Dass aerobe Bedingungen einen Einfluss auf das Auslösen der Expression von PaoABC haben, wurde in dieser Arbeit ebenfalls ermittelt.
Im Zusammenhang mit der Produktion von ROS durch PaoABC wurde die Funktion eines kürzlich in Elektronentransfer-Distanz zum FAD identifizierten [4Fe4S]-Clusters untersucht. Ein Austausch der für die Bindung des Clusters zuständigen Cysteine führte zur Instabilität der Proteinvarianten, weswegen für diese keine weiteren Untersuchungen erfolgten. Daher wird zumindest ein struktur-stabilisierender Einfluss des [4Fe4S]-Clusters angenommen. Zur weiteren Untersuchung der Funktion dieses Clusters, wurde ein zwischen FAD und [4Fe4S]-Cluster lokalisiertes Arginin gegen ein Alanin ausgetauscht. Diese Proteinvariante zeigte eine reduzierte Geschwindigkeit der Reaktion gegenüber dem Wildtyp. Die Bildung von Superoxid konnte auch hier nicht beobachtet werden. Die Vermutung, dass dieser Cluster einen elektronen-sammelnden Mechanismus unterstützt, welcher die Radikalbildung verhindert, kann trotz allem nicht ausgeschlossen werden. Da im Umkreis des Arginins weitere geladene und aromatische Aminosäuren lokalisiert sind, können diese den notwendigen Elektronentransfer übernehmen.
Neben der Ermittlung eines physiologischen Elektronenakzeptors und dessen Einfluss auf die Expression von PaoABC zeigt diese Arbeit auch, dass die Chaperone PaoD und MocA während der Reifung des MCD-Kofaktor eine gemeinsame Bindung an PaoABC realisieren. Es konnte im aktiven Zentrum von PaoABC ein Arginin beschrieben werden, welches auf Grund der engen Nachbarschaft zum MCD-Kofaktor und zum Glutamat (PaoABC-EC692) am Prozess der Substratbindung beteiligt ist. Im Zusammenhang mit dem Austausch dieses Arginins gegen ein Histidin oder ein Lysin wurden die Enzymspezifität und der Einfluss physiologischer Bedingungen, wie pH und Ionenstärke, auf die Reaktion des Enzyms untersucht. Gegenüber dem Wildtyp zeigten die Varianten mit molekularem Sauerstoff eine geringere Affinität zum Substrat aber auch eine höhere Geschwindigkeit der Reaktion. Vor allem für die Histidin-Variante konnte im gesamten pH-Bereich ein instabiles Verhalten bestimmt werden. Der Grund dafür wurde durch das Lösen der Struktur der Histidin-Variante beschreiben. Durch den Austausch der Aminosäuren entfällt die stabilisierende Wirkung der delokalisierten Elektronen des Arginins und es kommt zu einer Konformationsänderung im aktiven Zentrum.
Neben der Reaktion von PaoABC mit einer Vielzahl aromatischer Aldehyde konnte auch der Umsatz von Salicylaldehyd zu Salicylsäure durch PaoABC in einer Farbreaktion bestimmt werden. Durch Ausschluss von molekularem Sauerstoff als terminaler Elektronenakzeptor, in einer enzym-gekoppelten Reaktion, erfolgte ein Elektronentransport auf Ferrocencarboxylsäure. Die Kombination aus beiden Methoden ermöglichte eine Verwendung von Ferrocen-Derivaten zur Generierung einer enzym-gekoppelten Reaktion mit PaoABC.
Die Untersuchungen zu PaoABC zeigen, dass die Vielfalt der durch das Enzym katalysierten Rektionen weitere Möglichkeiten der enzymatischen Bestimmung biokatalytischer Prozesse bietet.
The facilitation of species coexistence has been a central theme in ecological research for years, highlighting two key aspects: ecological niches and competition between species. According to the competitive exclusion principle, the overlap of species niches predicts the amount of shared resources and therefore competition between species, determining their ability to coexist. Only if niches of two species are sufficiently different, thus niche overlap is low, competition within species is higher than competition between species and stable coexistence is possible. Thereby, differences in species mean traits are focused on and conspecific individuals are assumed to be interchangeable. This approach might be outdated since behaviour, as a key aspect mediating niche differentiation between species, is individual based. Individuals from one species consistently differ across time and situations in their behavioural traits. Causes and consequences of consistent behavioural differences have been thoroughly investigated stimulating their recent incorporation into ecological interactions and niche theory. Spatial components have so far been largely overlooked, although animal movement is strongly connected to several aspects of ecological niches and interactions between individuals. Furthermore, numerous movement aspects haven been proven to be crucially influenced by consistent individual differences. Considering spatial parameters could therefore crucially broaden our understanding of how individual niches are formed and ecological interactions are shaped. Furthermore, extending established concepts on species interactions by an individual component could provide new insights into how species coexistence is facilitated and local biodiversity is maintained.
The main aim of this thesis was to test whether consistent inter-individual differences can facilitate the coexistence of ecological similar species. Therefore, the effects of consistent inter-individual differences on the spatial behaviour of two rodent species, the bank vole (Myodes glareolus) and the striped field mouse (Apodemus agrarius), were investigated and put in the context of: (i) individual spatial niches, (ii) interactions between species, and (iii) the importance of different levels of behavioural variation within species for their interactions. Consistent differences of study animals in boldness and exploration were quantified with the same tests in all presented studies and always combined with observations of movement and space use via automated VHF radio telemetry. Consequently, results are comparable throughout the thesis and the methods provide a common denominator for all chapters. The first two chapters are based on observations of free-ranging rodents in natural populations, while chapter III represents an experimental approach under semi-natural conditions.
Chapter I focusses on the effect of consistent differences in boldness and exploration on movement and space use of bank voles and their contribution to individual spatial niche separation. Results show boldness to be the dominating predictor for spatial parameters in bank voles. Irrespective of sex, bolder individuals had larger home ranges, moved longer distances, had less spatial interactions with conspecifics and occupied different microhabitats compared to shy individuals. The same boldness-dependent spatial patterns could be observed in striped field mice which is reported in chapter II. Therefore, both study species showed individual spatial niche occupation.
Chapter II builds on findings from the first chapter, investigating the effect of boldness driven individual spatial niche occupation on the interactions between species. Irrespective of species and sex, bolder individuals had more interspecific spatial interactions, but less intraspecific interactions, compared to shy individuals. Due to individual niches occupation the competitive environment individuals experience is not random. Interactions are restricted to individuals of similar behavioural type with presumably similar competitive ability, which could balance differences on the species level and support coexistence.
In chapter III the experimental populations were either comprised of only shy or only bold bank voles, while striped field mice varied, creating either a shy- or bold-biased competitive community. Irrespective of behavioural type, striped field mice had more intraspecific interactions in bold-biased competitive communities. Only in a shy-biased competitive community, bolder striped field mice had less interspecific interactions compared to shy individuals. Bank voles showed no difference in intra- or interspecific interactions between populations. Chapter III highlights, that not only consistent inter-individual differences per se are important for interactions within and between species, but also the amount of behavioural variation within coexisting species.
Overall, this thesis highlights the importance of considering consistent inter-individual differences in a spatial context and their connection to individual spatial niche occupation, as well as the resulting effects on interactions within and between species. Individual differences are discussed in the context of similarity of individuals, individual and species niche width, and individual and species niche overlap. Thereby, this thesis makes one step further from the existing research on individual niches towards integrating consistent inter-individual differences into the larger framework of species coexistence.
Determining the relationship between genotype and phenotype is the key to understand the plasticity and robustness of phenotypes in nature. While the directly observable plant phenotypes (e.g. agronomic, yield and stress resistance traits) have been well-investigated, there is still a lack in our knowledge about the genetic basis of intermediate phenotypes, such as metabolic phenotypes. Dissecting the links between genotype and phenotype depends on suitable statistical models. The state-of-the-art models are developed for directly observable phenotypes, regardless the characteristics of intermediate phenotypes. This thesis aims to fill the gaps in understanding genetic architecture of intermediate phenotypes, and how they tie to composite traits, namely plant growth. The metabolite levels and reaction fluxes, as two aspects of metabolic phenotypes, are shaped by the interrelated chemical reactions formed in genome-scale metabolic network. Here, I attempt to answer the question: Can the knowledge of underlying genome-scale metabolic network improve the model performance for prediction of metabolic phenotypes and associated plant growth? To this end, two projects are investigated in this thesis. Firstly, we propose an approach that couples genomic selection with genome-scale metabolic network and metabolic profiles in Arabidopsis thaliana to predict growth. This project is the first integration of genomic data with fluxes predicted based on constraint-based modeling framework and data on biomass composition. We demonstrate that our approach leads to a considerable increase of prediction accuracy in comparison to the state-of-the-art methods in both within and across environment predictions. Therefore, our work paves the way for combining knowledge on metabolic mechanisms in the statistical approach underlying genomic selection to increase the efficiency of future plant breeding approaches. Secondly, we investigate how reliable is genomic selection for metabolite levels, and which single nucleotide polymorphisms (SNPs), obtained from different neighborhoods of a given metabolic network, contribute most to the accuracy of prediction. The results show that the local structure of first and second neighborhoods are not sufficient for predicting the genetic basis of metabolite levels in Zea mays. Furthermore, we find that the enzymatic SNPs can capture most the genetic variance and the contribution of non-enzymatic SNPs is in fact small. To comprehensively understand the genetic architecture of metabolic phenotypes, I extend my study to a local Arabidopsis thaliana population and their hybrids. We analyze the genetic architecture in primary and secondary metabolism as well as in growth. In comparison to primary metabolites, compounds from secondary metabolism were more variable and show more non-additive inheritance patterns which could be attributed to epistasis. Therefore, our study demonstrates that heterozygosity in local Arabidopsis thaliana population generates metabolic variation and may impact several tasks directly linked to metabolism. The studies in this thesis improve the knowledge of genetic architecture of metabolic phenotypes in both inbreed and hybrid population. The approaches I proposed to integrate genome-scale metabolic network with genomic data provide the opportunity to obtain mechanistic insights about the determinants of agronomically important polygenic traits.
Electrosynthesis and characterization of molecularly imprinted polymers for peptides and proteins
(2019)
Infection on the move
(2019)
Movement plays a major role in shaping population densities and contact rates among individuals, two factors that are particularly relevant for disease outbreaks. Although any differences in movement behaviour due to individual characteristics of the host and heterogeneity in landscape structure are likely to have considerable consequences for disease dynamics, these mechanisms are neglected in most epidemiological studies. Therefore, developing a general understanding how the interaction of movement behaviour and spatial heterogeneity shapes host densities, contact rates and ultimately pathogen spread is a key question in ecological and epidemiological research.
In my thesis, I address this gap using both theoretical and empirical modelling approaches. In the theoretical part of my thesis, I investigated bottom-up effects of individual movement behaviour and landscape structure on host density, contact rates, and ultimately disease dynamics. I extended an established agent-based model that simulates ecological and epidemiological key processes to incorporate explicit movement of host individuals and landscape complexity. Neutral landscape models are a powerful basis for spatially-explicit modelling studies to imitate the complex characteristics of natural landscapes. In chapter 2, the first study of my thesis, I introduce two complementary R packages, NLMR and landscapetools, that I have co-developed to simplify the workflow of simulation and customization of such landscapes. To demonstrate the use of the packages I present a case study using the spatially explicit eco-epidemiological model and show that landscape complexity per se increases the probability of disease persistence. By using simple rules to simulate explicit host movement, I highlight in chapter 3 how disease dynamics are affected by population-level properties emerging from different movement rules leading to differences in the realized movement distance, spatiotemporal host density, and heterogeneity in transmission rates. As a consequence, mechanistic movement decisions based on the underlying landscape or conspecific competition led to considerably higher probabilities than phenomenological random walk approaches due directed movement leading to spatiotemporal differences in host densities. The results of these two chapters highlight the need to explicitly consider spatial heterogeneity and host movement behaviour when theoretical approaches are used to assess control measures to prevent outbreaks or eradicate diseases.
In the empirical part of my thesis (chapter 4), I focus on the spatiotemporal dynamics of Classical Swine Fever in a wild boar population by analysing epidemiological data that was collected during an outbreak in Northern Germany persisting for eight years. I show that infection risk exhibits different seasonal patterns on the individual and the regional level. These patterns on the one hand show a higher infection risk in autumn and winter that may arise due to onset of mating behaviour and hunting intensity, which result in increased movement ranges. On the other hand, the increased infection risk of piglets, especially during the birth season, indicates the importance of new susceptible host individuals for local pathogen spread. The findings of this chapter underline the importance of different spatial and temporal scales to understand different components of pathogen spread that can have important implications for disease management.
Taken together, the complementary use of theoretical and empirical modelling in my thesis highlights that our inferences about disease dynamics depend heavily on the spatial and temporal resolution used and how the inclusion of explicit mechanisms underlying hosts movement are modelled. My findings are an important step towards the incorporation of spatial heterogeneity and a mechanism-based perspective in eco-epidemiological approaches. This will ultimately lead to an enhanced understanding of the feedbacks of contact rates on pathogen spread and disease persistence that are of paramount importance to improve predictive models at the interface of ecology and epidemiology.
Mouse aldehyde oxidases (mAOXs) have a homodimeric structure and belong to xanthine oxidase family of molybdo-flavoenzymes. In general, each dimer is characterized by three subdomains: a 20 kDa N-terminal 2x[2Fe2S] cluster containing domain, a 40 kDa central FAD-containing domain and an 85 kDa C-terminal molybdenum cofactor (Moco) containing domain. Aldehyde oxidases have a broad substrate specificity including the oxidation of different aldehydes and N-heterocyclic compounds. AOX enzymes are present in mainly all eukaryotes. Four different homologs of AOX were identified to be present with varying numbers among species and rodents like mice and rats contain the highest number of AOX isoenzymes. There are four identified homologs in mouse named mAOX1, mAOX3, mAOX2, and mAOX4. The AOX homologs in mice are expressed in a tissue-specific manner. Expression of mAOX1 and mAOX3 are almost superimposable and predominantly synthesized in liver, lung, and testis. The richest source of mAOX4 is the Harderian gland, which is found within the eye's orbit in tetrapods. Expression of mAOX2 is strictly restricted to the Bowman’s gland, the main secretory organ of the nasal mucosa.
In this study, the four catalytically active mAOX enzymes were expressed in a heterologous expression system in Escherichia coli and purified in a catalytically active form. Thirty different structurally related aromatic, aliphatic and N-heterocyclic compounds were used as substrates, and the kinetic parameters of all four mAOX enzymes were directly compared. The results showed that all enzymes can catalyze a broad range of substrates. Generally, no major differences between mAOX1, mAOX3 and mAOX2 were identified and the substrate specificity of mAOX1, mAOX3, and mAOX2 was broader compared to that of mAOX4 since mAOX4 showed no activity with substrates like methoxy-benzaldehydes, phenanthridine, N1-methyl-nicotinamide, and cinnamaldehyde and 4-(dimethylamino)cinnamaldehyde.
We investigated differences at the flavin site of the mAOX enzymes by measuring the ability of the four mAOX enzymes to oxidize NADH in the absence of oxygen. NADH was able to reduce only mAOX3. The four mouse AOXs are also characterized by quantitative differences in their ability to produce superoxide radicals. mAOX2 is the enzyme generating the largest rate of superoxide radicals of around 40% in relation to moles of substrate converted and it is followed by mAOX1 with a ratio of 30%.
To understand the factors that contribute to the substrate specificity of mAOX4, site-directed mutagenesis was applied to substitute amino acids in the substrate-binding funnel by the ones present in mAOX1, mAOX3, and mAOX2. The amino acids Val1016, Ile1018 and Met1088 were selected as targets. An increase in activity was obtained by the amino acid exchange M1088V in the active site identified to be specific for mAOX4, to the amino acid identified in mAOX3.
STERILE APETALA (SAP) is known to be an essential regulator of flower development for over 20 years. Loss of SAP function in the model plant Arabidopsis thaliana is associated with a reduction of floral organ number, size and fertility. In accordance with the function of SAP during early flower development, its spatial expression in flowers is confined to meristematic stages and to developing ovules. However, to date, despite extensive research, the molecular function of SAP and the regulation of its spatio-temporal expression still remain elusive.
In this work, amino acid sequence analysis and homology modeling revealed that SAP belongs to the rare class of plant F-box proteins with C-terminal WD40 repeats. In opisthokonts, this type of F-box proteins constitutes the substrate binding subunit of SCF complexes, which catalyze the ubiquitination of proteins to initiate their proteasomal degradation. With LC-MS/MS-based protein complex isolation, the interaction of SAP with major SCF complex subunits was confirmed. Additionally, candidate substrate proteins, such as the growth repressor PEAPOD 1 and 2 (PPD1/2), could be revealed during early stages of flower development. Also INDOLE-3-BUTYRIC ACID RESPONSE 5 (IBR5) was identified among putative interactors. Genetic analyses indicated that, different from substrate proteins, IBR5 is required for SAP function. Protein complex isolation together with transcriptome profiling emphasized that the SCFSAP complex integrates multiple biological processes, such as proliferative growth, vascular development, hormonal signaling and reproduction. Phenotypic analysis of sap mutant and SAP overexpressing plants positively correlated SAP function with plant growth during reproductive and vegetative development.
Furthermore, to elaborate on the transcriptional regulation of SAP, publicly available ChIP-seq data of key floral homeotic proteins were reanalyzed. Here, it was shown that the MADS-domain transcription factors APETALA 1 (AP1), APETALA 3 (AP3), PISTILLATA (PI), AGAMOUS (AG) and SEPALLATA 3 (SEP3) bind to the SAP locus, which indicates that SAP is expressed in a floral organ-specific manner. Reporter gene analyses in combination with CRISPR/Cas9-mediated deletion of putative regulatory regions further demonstrated that the intron contains major regulatory elements of SAP in Arabidopsis thaliana.
In conclusion, these data indicate that SAP is a pleiotropic developmental regulator that acts through tissue-specific destabilization of proteins. The presumed transcriptional regulation of SAP by the floral MADS-domain transcription factors could provide a missing link between the specification of floral organ identity and floral organ growth pathways.