Here, we explored this idea by analyzing a selected group of putative σ54 promoters identified in Myxococcus xanthus natural product gene clusters. We show that mutations in putative σ54-RNA polymerase binding regions and in putative Nla28 EBP binding sites dramatically reduce in vivo promoter activities in growing and developing cells. We also show in vivo promoter activities are reduced in a nla28 mutant, that Nla28 binds to wild-type fragments of these promoters in vitro, and that in vitro binding is lost when the Nla28 binding sites are mutated. Together, our results indicate that M. xanthus uses σ54 promoters for transcription of at least some of its natural product genes. Interestingly, the vast majority of experimentally confirmed and putative σ54 promoters in M. xanthus natural product loci are located within genes and not in intergenic sequences.Bacterial-derived polyketide and non-ribosomal peptide natural products are crucial sources of therapeutics and yet little is known about the conditions that favor activation of natural product genes or the regulatory machinery controlling their transcription. Recent findings suggest that the σ One crucial component of this regulatory system is the σ54 protein, which directs RNA polymerase to conserved DNA sequences located in the − 12 and − 24-bp regions of σ54 promoter elements2. Enhancer binding proteins (EBPs), which are transcriptional activators, are also crucial for the normal function of the σ54 regulatory system. Namely, EBPs are ATPases that use the energy from ATP hydrolysis to help σ54-RNA polymerase form an open promoter complex and initiate transcription5. Bacteria typically have one gene for σ54, but often have multiple genes for EBPs; each EBP works with σ54 to regulate a subset of σ54 promoters, which the EBP identifies via specific tandem repeat sequences or enhancer elements7. Interestingly, the tandem repeat binding sites of EBP dimers are typically located 80- to 150-bp upstream of the − 12 and − 24-bp regions of σ54 promoters; hence, it seems likely that many σ54 promoters have intrinsically curved DNA sequences or binding sequences for DNA bending proteins, as EBP dimers directly contact σ54-RNA polymerase10.The σ7. Typically, the N-terminal signaling domain modulates the ATPase activity of the EBP in response to an intracellular or extracellular signal. In some cases, the N-terminal domain binds directly to a signaling molecule. However, the N-terminal domain of most EBPs is modified  by a signal transduction partner such as a histidine protein kinase that detects the signal11. Because σ54-RNA polymerase requires the energy from EBP-catalyzed ATP hydrolysis to initiate transcription and the EBP’s ATPase activity is controlled by signal input, the σ54 system is able to tightly control transcription of its target genes.EBPs generally contain three domains: an N-terminal signaling domain, a central ATPase domain that is responsible for ATP hydrolysis and transcriptional activation, and a C-terminal DNA-binding domain (DBD) that recognizes a specific DNA sequence54 was viewed as specialized regulatory system that was mainly dedicated to transcription of genes involved in nitrogen assimilation or nitrogen fixation13. In recent years however, it has become clear that the σ54 system is important for transcription of many types of bacterial genes. For example, the σ54 system in Bacillus cereus, Pseudomonas aeruginosa and Escherichia coli modulates transcription of genes involved amino acid metabolism, the response to reactive nitrogen species and the phage shock response, respectively16. In Caulobacter crescentus, Pseudomonas aeruginosa, Pseudomonas putida and Vibrio cholerae the σ54 system regulates genes that are important for flagellar biosynthesis and motility21, and in Pseudomonas aeruginosa the σ54 system is implicated in transcription of genes involved in quorum sensing, biofilm formation and virulence24.Historically, σ54 system in the soil bacterium Myxococcus xanthus has been studied extensively and has some rare properties. Namely, M. xanthus is one of the rare bacterial species in which the σ54 system has been linked to growth in nutrient rich conditions. For example, inactivation of the nla4 or nla18 EBP gene severely impairs M. xanthus growth in nutrient rich media27. Presumably, the relatively slow growth of the nla4 mutant and nla18 mutant is due at least in part to changes in the basal levels of the intracellular starvation signal (p)ppGpp and the resulting changes in gene expression.The σM. xanthus also has an unusually large repertoire of 53 EBP genes28. All of the EBPs were characterized a number of years ago and many of the EBPs were implicated in motility30 and in starvation-induced biofilm formation36, which yields spore-filled aerial structures called fruiting bodies. Six of the EBPs that begin functioning in the early to middle stages of biofilm development, which is also known as fruiting body development, form a regulatory cascade30. This EBP cascade is reminiscent of the sigma factor cascade that controls the sequential stages of spore development in Bacillus subtilis37, as pairs of EBPs functioning at one stage of development directly activate transcription of an EBP gene important for the next developmental stage.39. The consensus Nla28 binding site [CT(C/G)CG(C/G)AG consensus half site], which was generated from these studies, was subsequently used to search the M. xanthus genome sequence for Nla28 target promoters/genes located outside the EBP cascade39. A number of these putative Nla28 target promoters were identified in natural product gene clusters. This was an intriguing finding, as Volz et al. previously showed that two M. xanthus EBPs (HsfA and MXAN4899) are capable of binding to fragments of natural product gene promoters40. Furthermore, it was previously suggested that the σ54 system might be a key regulator of polyketide (PK) and non-ribosomal peptide (NRP) natural product genes in M. xanthus, and in bacteria in general, based on bioinformatics38.Nla28 is one of the early-functioning developmental EBPs that participates in the transcriptional cascade. A putative tandem repeat promoter binding site for Nla28 dimers was identified and analyzed using bioinformatics and experimentation54 system modulates expression of M. xanthus natural product genes. We focused on three potential σ54 promoter targets of Nla28 that are located in M. xanthus natural product gene clusters38. The activities of the three natural product promoters increase during growth, with peak activities occurring during the transition into stationary phase. The activities of the promoters also increase during starvation-induced development. These findings are consistent previous studies linking expression of bacterial natural product genes to changes in nutritional status45. Our in vivo and in vitro mutational analyses indicate that three natural product promoters are indeed σ54 promoter elements and direct targets of Nla28, as predicted via bioinformatics. An examination of the characterized and uncharacterized σ54 promoters in M. xanthus natural product gene clusters yielded a striking result; the vast majority are predicted to be intragenic not intergenic promoters. These results are consistent with previous experimental findings, which placed many characterized M. xanthus σ54 promoters in intragenic regions39.Here, we explored the idea that the σM. xanthus strains were grown at 32 °C in CTTYE broth  or on CTTYE plates containing 1.5% agar. Fifty μg/ml of kanamycin or 10 μg/ml of tetracycline were added to CTTYE broth and CTTYE agar plates as needed. CTT soft agar (CTTSA), which is used to plate electroporated M. xanthus cells, contains 1% Casitone, 10 mM Tris (pH 8.0), 1.0 mM KH2PO4 (pH 7.6), 8.0 mM MgSO4, and 0.7% agar. Submerged culture development of M. xanthus strains occurred in 24-well polystyrene plates containing 100 μl of MC7 buffer . Unless otherwise stated, E. coli strains were grown in Luria–Bertani (LB) broth  supplemented with 100 μg/ml of ampicillin.Bacterial strains and plasmids used in the study are listed in Table M. xanthus strains were grown by inoculating cells into flasks containing CTTYE broth and incubating the cultures at 32 °C with vigorous swirling. Development was induced as previously described25. Briefly, M. xanthus cells were grown in CTTYE broth until the cultures reached a density of approximately 5 × 108 cells/ml, the cells were pelleted, the supernatant was removed, and the cells were resuspended in MC7 buffer to a density of 5 × 109 cells/ml. Forty μl aliquots of the cell suspensions were placed into polystyrene plate wells containing 100 μl of MC7 buffer and the polystyrene plates were transferred to a 32 °C incubator for 24 h.M. xanthus strain DK162246 was extracted using ZYMO Research gDNA extraction kit. Oligonucleotides used in PCR reactions were synthesized by Integrated DNA Technologies (IDT) and are listed in Table Chromosomal DNA from wild-type 54 promoter elements were generated using the Quick Lightning Mutagenesis Kit from Agilent Technologies and the manufacturer’s protocol. Briefly, promoter fragments containing the putative σ54-RNA polymerase binding site in the − 12 and − 24-bp regions and the upstream Nla28 tandem repeat binding site were cloned into pCR 2.1 TOPO vector (Invitrogen). Mutations in the − 12-bp region, the − 24-bp region, the spacer between the − 12 and − 24-bp regions were generated using primers carrying the appropriate nucleotide changes  were synthesized by IDT; the first putative Nla28 half binding site in each promoter was changed to AAAAAAAA. The mutant promoter fragments were then subcloned into the promoterless lacZ expression vector pREG172747, introduced into M. xanthus strains and analyzed as described below.Site-directed mutations in putative σes Table , plasmidlacZ expression vector pREG1727 to create lacZ transcriptional fusions47. The plasmids were introduced into strain DK1622 or a derivative of strain DK1622 carrying an insertion in the nla28 gene, and cells carrying a plasmid integrated at the Mx8 phage attachment site in the chromosome were identified via PCR. The in vivo activities of wild-type and mutant promoters were determined by measuring the specific activities of β-galactosidase in cells developing in submerged cultures for 1, 2, 6, 12 or 24 h, or growing in CTTYE broth for various amounts of time49. Mean wild-type and mutant promoter activities were compared using a two-way analysis of variance (ANOVA) and Tukey’s multiple comparisons post hoc tests, considering two independent variables in each group. The significance level was set at p < 0.05 or lower. GraphPad Prism software v8.4  was used for all analyses.Wild-type and mutant MXAN1286, MXAN1579, MXAN1603 and MXAN3778 promoter fragments were cloned into the promoterless nla28 gene corresponding to the Nla28 DNA binding domain (Nla28-DBD)30 was PCR amplified using gene-specific primers (Table E. coli strain BL21 (DE3) using chemical transformation. Cells containing the Nla28-DBD expression plasmids were grown in rich LB broth to a density of 2 × 108 cells/ml. Protein expression was induced by the addition of 0.3 mM IPTG to the culture and the subsequent incubation of the culture for 12 h at 15 °C. Cells were pelleted via centrifugation and resuspended in 25 ml column buffer  per liter of culture. The resuspended cells were lysed by a combination of freeze-thawing and sonication, and pelleted by centrifugation. The crude extract (supernatant) containing Nla28-DBD was diluted by adding 125 ml of cold column buffer to every 25 ml aliquot of crude extract. For purification of Nla28-DBD from the diluted crude extract, 15 ml of amylose resin was placed in a 2.5 × 10 cm column and the amylose resin was washed with 250 ml cold column buffer. Diluted crude extract containing Nla28-DBD was loaded onto amylose columns at a flow rate of 5 ml/min and washed with 600 ml cold column buffer at a flow rate of 10 ml/min. Nla28-DBD was eluted using 100 ml cold column buffer containing 10 mM maltose; the flow rate was 5 ml/min and 20 fractions containing 5 ml were collected. The presence of eluted Nla28-DBD was detected by UV absorbance at 280 nm. Nla28-DBD- containing fractions were pooled and incubated with 1 mg of Factor Xa at 4 °C overnight to cleave the MBP tag. Nla28-DBD was separated from MBP and concentrated to about 1 mg/ml using Amicon Ultra centrifugal filter units (EMD Millipore). SDS-PAGE and Bradford assays were used to determinate the purity and concentration of Nla28-DBD.A fragment of the rs Table , and the1286, Cy5-P1579 and Cy5-P3778) were generated via PCR; each promoter fragment contained a putative wild-type Nla28 binding site. Three mutant derivatives of these 5′ Cy5-labelled promoter fragments  were synthesized; the first putative Nla28 half binding site in each promoter was changed to AAAAAAAA. All PCR-generated and synthesized promoter fragments were gel-purified and used in subsequent EMSAs. In EMSA reactions, 2 μM purified Nla28-DBD was incubated with 1 ng of 5′ Cy5-labelled wild-type promoter fragment  or 5′ Cy5-labelled mutant promoter fragment  in EMSA buffer  for 30 min at 30 °C. The samples were then analyzed using PAGE under non-denaturing conditions and imaged using a Bio-Rad imager.Purified Nla28-DBD was expected to bind to wild-type MXAN1286, MXAN1579 and MXAN3778 promoter fragments carrying a putative Nla28 tandem repeat binding site. Using the 5′ Cy5-labelled oligonucleotides shown in Table 38, the algorithm developed by Studholme et al.50 was used to examine whether the σ54 system might be a common regulator of bacterial natural product genes. Namely, 180 annotated PK and NRP gene clusters from 58 bacterial species were analyzed for sequences that closely match the σ54 promoter consensus in the − 12-bp region and in the − 24-bp region . The results, which uncovered 124 clusters with at least one σ54 promoter based on consensus matching, supported the idea that the σ54 system might be a general regulator of bacterial natural product genes.In a previous studyM. xanthus) uses the σ54 system for transcription of PK and NRP gene clusters, as predicted in the bioinformatics analysis of Stevens et al.38. We focused on the putative σ54 promoters upstream of MXAN1286, MXAN1579, MXAN1603 and MXAN3778, as these genes were linked to natural product biosynthetic genes/clusters. Namely, the MXAN1286 gene is embedded in a putative NRP gene cluster and flanked by genes that are likely to be involved in NRP biosynthesis. MXAN1579 and MXAN1603 are predicted to be part of another M. xanthus NRP gene cluster. MXAN1603 is a putative non-ribosomal peptide synthetase gene. MXAN1579 is embedded in the cluster and flanked by genes such as MXAN1603 that are likely to be involved in NRP biosynthesis. MXAN3778 is adjacent to a putative hybrid non-ribosomal peptide synthetase/type I polyketide synthase gene and possibly part of the same operon38.The goal of the work presented here was to examine whether a major producer of bacterial natural products ] and the distance from putative Nla28 binding sites2. The mutations that we generated are the following: the GC dinucleotide in the − 12-bp region was replaced with a TT, the GG (GA in MXAN3778) dinucleotide in the − 24-bp region was replaced with a TT, or 1 bp in the spacer between the − 12 and − 24-bp regions was deleted. Subsequently, wild-type and mutant promoter fragments were fused to the promoterless lacZ gene in plasmid pREG172747 and the lacZ transcriptional fusion plasmids were introduced into wild-type M. xanthus strain DK1622 (the plasmids integrated at the Mx8 phage attachment site in the chromosome).To confirm that the natural product loci have bona fide σon Table . HoweverlacZ expression. As shown in Fig. 54-RNA polymerase binding substantially impacted the activities of the MXAN1286, MXAN1579 and MXAN3778 promoters in growing and developing cells, supporting the prediction that the three natural product loci use σ54 promoter elements for transcription.Wild-type and mutant promoter activities during growth in CTTYE broth and fruiting body development in MC7 starvation buffer were inferred from the levels of 54-RNA polymerase binding site in the MXAN1603 promoter region only had a modest impact on growth-related and developmental activities. Our interpretation of this result is that the fragment of the MXAN1603 promoter region contains an unidentified promoter element that is responsible for the majority of the observed development and growth-related activities and that the putative σ54-type promoter element only makes a minor contribution to the observed activities.In contrast to the other promoters, the MXAN1603 promoter only showed a slight increase (about 1.3-fold) in activity during growth in CTTYE Figure A and dur54 promoters, as EBP-mediated ATP hydrolysis opens the σ54-RNA polymerase promoter complex so that transcription can initiate5. Since the σ54 promoters in the MXAN1286, MXAN1579, and MXAN3778 loci were identified as potential targets of Nla28, we determined whether the activities of wild-type promoter fragments are reduced as predicted in a mutant containing an inactivated nla28 gene25 . MXAN1286, MXAN1579, and MXAN3778 promoter activities in wild-type and nla28 mutant cells grown in CTTYE broth are shown in Fig. nla28 abolished the growth phase regulation of all three promoters and caused about 3.1- to fourfold reduction in peak promoter activities at the highest cell densities. Inactivation of nla28 also abolished the developmental activities of the promoters, as the promoters did not show the typical increases in activities when nla28 cells were placed in MC7 starvation buffer  Nla28 half binding site in each promoter fragment was converted to all A nucleotides. Wild-type and mutant promoters were introduced into wild-type strain DK1622 and promoter activities during growth in CTTYE broth and development in in MC7 starvation buffer were determined CG(C/G)AG], in the σsee Fig. . To exam54 promoter elements of the MXAN1286, MXAN1579 and MXAN3778 natural product loci and that the tandem repeats that we identified in the σ54 promoter elements are Nla28 binding sites.Electrophoretic mobility shift assays (EMSAs) were used to confirm that the MXAN1286, MXAN1579, and MXAN3778 natural product promoters are targets of the Nla28 EBP. In particular, we used EMSAs to determine whether the purified DNA binding domain of Nla28 (Nla28-DBD) is capable of binding a fragment of the MXAN1286 promoter, MXAN1579 promoter, and MXAN3778 promoter. Each promoter fragment, which corresponded to DNA upstream of − 12 and − 24-bp regions, contained a putative binding site for a Nla28 dimer. As shown in Fig. M. xanthus EBPs (HsfA and MXAN4899) are capable of binding to fragments of natural product gene promoters40. With this information and the preliminary data from Nla28 studies in mind, Stevens et al. asked if the σ54 system might be a common regulator natural product genes38. Namely, a bioinformatics analysis was used to search for putative σ54 promoters in the sequences of 180 PK or NRP gene clusters from 58 bacterial species. The results, which revealed that about 70% of natural product gene clusters have at least one putative σ54 promoter, suggested that the σ54 system might indeed be a common regulator of natural product genes.For decades, bacterial-derived PK and NRP natural products have been a crucial source of therapeutic agents such as antibiotics and yet little information about the regulation of these genes has been uncovered. In a notable study in 2012, Volz et al. showed that two M. xanthus is an excellent system to study natural product gene regulation, as this bacterium is a major producer of PKs and NRPs and over 80 putative σ54 promoters were identified in the PK and NRP gene clusters of strain DK162238  contain bona fide σ54 promoter elements and that the σ54 promoters are targets of the EBP Nla28. Our results also indicate that the σ54 promoters are crucial for both growth-related and developmental activities. We propose that the fourth natural product promoter region that we characterized, MXAN1603, also contains a σ54 promoter. However, it seems likely that the MXAN1603 promoter region contains an unidentified promoter element that is responsible for the majority of the observed developmental and growth-related activities and the σ54-type promoter only makes a minor contribution to these activities.Our in vivo and in vitro data indicate that three of the 55. Nla28 begins modulating gene expression in the early stages of starvation-induced fruiting body development39, which led to the suggestion that the Nla28/Nla28S signal transduction system might be a general regulator of starvation-induced or stress-responsive genes55. The findings presented here are consistent with this idea. In particular, the MXAN1286, MXAN1579, and MXAN3778 σ54 promoters are induced in the early-middle stages of fruiting body development and the developmental activities of these promoters are dependent on Nla28 ppGpp accumulates when M. xanthus transitions from exponential growth to stationary phase, since this seems to be the case in other bacterial species58. In addition, bacterial natural product gene expression was previously linked to (p)ppGpp accumulation59 and stringent response-associated genes such as relA and spoT have been linked to synthesis of bacterial natural products60.The above results point to nutrient depletion/starvation as the potential trigger for Nla28/Nla28S-mediated activation of the MXAN1286, MXAN1579 and MXAN3778 promoters. We suggest that the stringent response and accumulation of (p)ppGpp might also be a key inducing factor, as accumulation of this starvation signal is known to initiate 54 promoters that we characterized are located within the coding sequences of genes (intragenic) and not in intergenic regions , the σ54 promoter is located in the coding sequence of an upstream gene . The question that arises is, why would a σ54 promoter be located so far upstream of the single gene or operon whose transcription it aims to regulate? One possibility is that the natural product σ54 promoter is positioned at a distant location to produce a relatively long 5′ untranslated region (UTR) in the mRNA and that the 5′ UTR provides an additional layer of regulation for natural product gene expression. We should note that a substantial number of the internal σ54 promoters are predicted to yield relatively long 5′ UTRs as well. Why would many natural product genes need multiple levels of regulation? Natural product synthesis is often energy expensive and many natural products are toxic at high levels; hence, tight and signal-responsive regulation of natural product gene expression might be important in many cases.A second and relatively large group of putative natural product σ54-mediated regulation of natural product genes and other types of bacterial genes will require researchers to go beyond predicting where σ54 promoters are located. Indeed, we argue that the work of confirming putative σ54 promoters and analyzing the mRNAs under σ54 control is crucial, as the results will help address a number of long-held assumptions about bacterial promoters and bacterial gene regulation in general.Of course, addressing questions about the role and mechanism of σSupplementary Information


Additionally, a 10−6 mol·L−1 solution of lysozyme was successfully detected using the Mg–Au nanopore substrates. Our low-cost method is reproducible, homogeneous, and suitable for the fabrication of SERS substrates.Based on an electrochemical method, three-dimensional arrayed nanopore structures are machined onto a Mg surface. The structured Mg surface is coated with a thin gold (Au) film, which is used as a surface-enhanced Raman scattering (SERS) substrate. A rhodamine 6G (R6G) probe molecule is used as the detection agent for the SERS measurement. Different sizes of arrayed micro/nanostructures are fabricated by different treatment time using the electrochemical process. The topographies of these micro/nanostructures and the thickness of the Au film have an influence on the Raman intensity of the Mg substrate. Furthermore, when the thickness of Au film coating is held constant, the Raman intensity on the structured Mg substrates is about five times higher after a treatment time of 1 min when compared with other treatment times. The SERS enhancement factor ranges from 10 Surface-enhanced Raman spectroscopy (SERS) can be used to detect biomolecules –3, explo−4 mol·L−1) is then detected using the Klarite substrate. Candeloro et al. [−6 mol·L−1) were used with the substrate for detection. Au nanostructures of different shapes and sizes  have also been fabricated using EBL and reactive ion etching methods [5. Wu et al. [6 was achieved for 4-mercaptobenzoic acid molecules on the arrayed Au nanoholes. However, lithography-based methods have limitations, as they are inefficient and cannot be exploited for mass production. In addition, it is challenging to use the existing methods to fabricate more complex nanostructures.Recently, all kinds of shapes of nanostructures machined by several researchers as SERS substrates have been machined by using lithography-based method –20. Addio et al.  employed methods . The Ramu et al.  machined6 and 1.74 × 107. Gao et al. [−6 mol·L−1. Compared with other processing methods, the precision of FIB processing technology is relatively high. However, FIB processing is an expensive and low-throughput technology. In addition, the processing time of a wide range of micro/nanostructures is long. Therefore, the low-cost and efficient preparation of array nanostructures with controllable shape, size and density is urgently required for SERS substrates for molecular recognition.Focused ion beam (FIB) technology can also be used to directly fabricate high-precision nanostructures on surfaces made of silicon, silicon dioxide and metal –33. FIB 2 nanocomposites formed on Pt substrates yielded a SERS enhancement factor of 1.8 × 108 for R6G molecules [−13 mol·L−1 and the highest enhancement factor achieved was 2.3 × 108. Yang et al. [−8 mol·L−1. Based on a ORC method, Chen et al. [x with reverse rates of 200, 100, 50, 25, and 5 mV/s. The highest enhancement factor observed with R6G in this system was 5.5 × 106 with a reverse rate of 25 mV/s. Furthermore, pigments of Brilliant Blue FCF and Indigo Carmine at concentrations as low as 10−8 mol·L−1 and 10−7 mol·L−1, respectively, were detectable using the SERS substrate. Ou et al. [−6 mol·L−1) on the Ag substrate was larger with subsequent drying treatment than without.Some researchers have fabricated nanostructures as SERS substrates by using electrochemical oxidation–reduction cycle (ORC) methods –39. Geneolecules . Chang eolecules  fabricatg et al.  used ORCn et al.  created u et al.  prepared−9 mol·L−1 for R6G molecules was obtained using the aforementioned SERS substrates. Choi et al. [7 was obtained with an Au thickness of 20 nm and an AAO thickness of 100 nm. Using an AAO template, Aflatoxin B1 (AFB1) from peanut extract was detected at concentrations ranging from 1.5 µg/L to 1.5 mg/L. Although the method is suitable for the evaluation of AFB1 content in food safety inspections [Anodic aluminum oxide (AAO) has been used as a mask to fabricate nanodot SERS substrates –43. Usini et al.  used a nIn this paper, a simple and rapid electrochemical micromachining approach is presented for fabricating sensitive three-dimensional SERS substrates. First, by controlling the parameters of plasma electrolytic oxidation (PEO) treatment, arrayed nanopores were formed on a Mg surface. Then, the nanopore surfaces were coated by incubating them with Au films for different lengths of time. The nanostructures were fabricated by controlling the treatment time and R6G molecules were chosen to be adsorbed onto the substrate. Finally, the Raman intensities of low concentrations of lysozyme were determined using arrayed structures as the SERS substrates. Using this approach allows for the accurate quantification of extremely small amounts of protein.3) for anodic oxidation treatment. Prior to the treatment, all specimens were ground using SiC paper up to 1200 grit, and then degreased with ethanol and deionized water in succession. No further purification was carried out. A customized DC power supply was used to conduct the PEO. 3PO4 in deionized water. The PEO treatment was performed in constant-current mode with a fixed constant current density of 25 mA/cm2. The frequency and duty ratio were 500 Hz and 50%, respectively. The electrolyte temperature was regulated within 30 ± 2 °C by a mechanical stirring cooling system. To study the influence of surface conditions  on the SERS intensity, the duration of the PEO treatment was set as the single variable , as shown in As-cast Mg ingots were sliced into rectangular coupons (15 × 15 × 4 mm−7 mol·L−1) for 20 min. The excess R6G molecules were removed by rinsing with ethanol and a gentle nitrogen flow was used to dry the samples.The structured Mg surfaces were coated with Au of varying thickness and dipped into an aqueous solution of R6G  with 532 and 785 nm lasers was used. The incident optical power was kept at 0.6 mW with a 50× objective and the beam diameter was approx. 1 μm. The signal detector used a CCD camera (1040 × 256 pixels) with a grating size of 1800 lines/mm. The exposure time was 1 s and one accumulation scan was employed. The mapping images of the Raman spectrum were scanned over a 20 × 20 μm2. In addition, a scanning electron microscopy (SEM) system  was employed to characterize the nanopores.An atomic force microscopy (AFM) system  was employed to detect the two-dimensional and three-dimensional topographies of the nanopores. Imaging was performed in contact mode and an elastic constant of 0.2 N/m was selected for the silicon cantilever. The scanning area was 50 × 50 μmSurface roughness and chemical composition have a strong influence on the intensity of Raman signals. PEO was employed to fabricate a porous oxide layer on a Mg alloy surface, which benefits from the increase of surface roughness and shows the potential for storing micro- or nanoparticles. During PEO treatment, the intrinsic passivation layer of the Mg alloy is disrupted in random positions through local melting during electrical breakdowns. After cooling by the electrolyte, a stable oxide layer containing arrayed pores is deposited on the surface. The parameters and duration of the PEO process should be carefully determined, as intensive energy input and longer treatment duration may create excessive surface roughness or even introduce unexpected defects on the surface. Thus, a set of moderate parameters was applied in this work to ensure that the Raman signal reflection properties were optimal.Structurally similar nanopores can be machined by using mechanical machining methods. For instance, micro/nanostructures can be fabricated using diamond turning, such as single point diamond turning or diamond fly cutting. For example, sinusoidal grid surfaces can be formed with the aid of fast tool servos. Nanopores with depths of 6.33 µm can be generated by using diamond turning .The PEO treatment time and the thickness of the Au film can have a significant impact on the performance of the SERS substrate. The performance was quantified by measuring Raman enhancement, which was determined by using R6G as a probe molecule.The effect of Raman enhancement of R6G molecules with different thicknesses of the gold film on the same structure surface  was studied. The data of Raman mapping were exported from the Raman spectra point by point.−7 M) on nanopore substrates coated with different thicknesses of the Au film after a constant time of PEO treatment (5 min). The characteristic Raman peaks of R6G molecules were detected at 611, 772, 1183, 1311, 1362, 1503, and 1605 cm−1. The results indicate that the Raman signal was most intense when the nanopore substrate with a 10 nm thick Au film was used. The Raman intensity of R6G decreased as the thickness of the Au film increased. Accordingly, the Raman intensity of the R6G signal derived from substrates coated with a 30 nm thick Au film was very low, as shown in −7 mol·L−1) on nanopore structures that were fabricated using PEO different treatment times. The microstructures vary in their morphology depending to the length of the treatment time, which in turn has a profound effect on the Raman intensity. Compared with other structures generated using different treatment times, a bare surface did not yield a high Raman intensity when measuring R6G. The Raman intensity was strongest when surfaces were PEO-treated for 1 min and a Au film of 10 nm thickness was deposited  in ethanol solution on nanopore substrates that were fabricated with a PEO treatment time of 1 min and were coated with a Au film of 10 nm thickness. The characteristic Raman peaks of lysozyme molecules were detected at concentrations as low as 10−6 mol·L−1. These peaks included SS bridge (521 cm−1), phenylalanine (601 cm−1), tryptophan(s) (760 cm−1), tyrosine doublet (858 cm−1), tryptophan (881 cm−1), CC stretching (934 cm−1), tyrosine , tryptophan (1337 cm−1), COO− symmetric stretch(s) (1384 cm−1), and tryptophan (1554 cm−1). Together, these data show that the electrochemical method can be used to fabricate nanopores as SERS substrates for the sensitive detection of proteins such as lysozyme.6 and 1.75 × 107. Experiments with lysozyme demonstrated that the Mg–Au nanopore substrates can be used to detect low levels of proteins (10−6 mol·L−1). Due to its reliability, homogeneity, low cost and high sensitivity, the system described herein holds great promise for future protein detection and quantification applications.Two- and three-dimensional arrayed micro/nanopores can be machined on Mg substrates by using a novel electrochemical method. The optimal treatment time for the process was 1 min, and the SERS intensity of the R6G molecules was enhanced by applying a 10 nm Au film onto the structured Mg surface. The SERS enhancement factor of this optimized system was between 10


This giant positive expansion originates from the increased mobility of the dodecyl chains with temperature that determine the intermolecular interactions and the network spacing.Thermal expansion, the response in shape, area or volume of a solid with heat, is usually large in molecular materials compared to their inorganic counterparts. Resulting from the intrinsic molecule flexibility, conformational changes or variable intermolecular interactions, the exact interplay between these mechanisms is however poorly understood down to the molecular level. Here, we investigate the structural variations of a two-dimensional supramolecular network on Au(111) consisting of shape persistent polyphenylene molecules equipped with peripheral dodecyl chains. By comparing high-resolution scanning probe microscopy and molecular dynamics simulations obtained at 5 and 300 K, we determine the thermal expansion coefficient of the assembly of 980 ± 110 × 10 −6 K−1 is observed by scanning probe microscopy in a supramolecular network on a gold surface.The intrinsic flexibility of molecules opens the door to unusual physical properties. Now, a large thermal expansion coefficient of 980 ± 110 × 10 Typically, thermal expansion coefficients  of inorganic materials are in the range of 0–20 × 10−6 K−1, whereas molecular counterparts vary from 50–60 × 10−6 K−1 (poly(styrene))2 up to  ~420 × 10−6 K−1 for porous coordination polymers1. Compared to inorganic materials, molecular architectures enable larger positive (or even negative5) expansions due to various mechanisms involving the intrinsic molecules flexibility, their propensity to conformational changes or the weak intermolecular interactions 2. To date, however, understanding of the underlying mechanisms governing such large expansion coefficients at the molecular scale remains elusive.The expansion of a material upon heating, known as thermal expansion is a fundamental property of solid systems that governs many of their mechanical applications7, in the solid state or at the solid interfaces11. Resulting from weak non-covalent intermolecular interactions, supramolecular assemblies enable the growth of well-extended and highly-ordered structures with tunable chemical, optochemical or mechanical properties13. In aqueous environments, extended polycyclic hydrocarbon molecule (PAH) assemblies have been also reported through the interdigitation of long alkyl chains18. While these organic architectures have already shown potential applications in photovoltaics and nanoelectronics19, the weak intermolecular interactions governing these assemblies and the intrinsic flexibility of the precursors also offer new opportunities for the design of responsive materials to external stimuli20. For instance, the thermal response of 2D supramolecular assemblies has never been investigated down to the molecular scale.Supramolecular chemistry has opened these last years new avenues in controlling spontaneous bottom-up assemblies of molecules both in solution 25. The growth of two-dimensional supramolecular networks is nowadays successfully achieved at surfaces by the thermal evaporation of precursors in ultra high vacuum (UHV)26. This approach, however, limits the size of the precursors and prevents the use of fragile moieties such as long alkyl chains that might degrade during evaporation. In liquid, such size or composition limitations are not present18. In UHV conditions, the electrospray deposition (ESD) technique allows the introduction of large molecules. Therefore, it is possible to study in UHV conditions the adsorption properties of molecules and their assemblies that mimic established assemblies at the liquid–solid interface33. In this context, temperature-dependent STM/AFM investigations of supramolecular systems might enable the analysis of their thermoresponse down to the atomic scale.To address supramolecular systems at the nanoscale, atomic force microscopy (AFM) and scanning tunneling microscopy (STM) are suitable techniques since they allow the highest spatial resolutions of single molecules and their assemblies at surfaces−6 K−1, which is supported by microsecond long temperature-dependent MD simulations. The alkyl chains attached to the molecular cores are found to undergo large thermal fluctuations (compared to the core) as a result of entropic effects and large anharmonic vibrations. This results in temperature-dependent intermolecular interactions that promote the giant expansion of the highly-ordered supramolecular network with temperature.Here, we investigate the thermal response of a 2D supramolecular assembly composed of shape persistent spoked wheel molecules equipped with peripheral dodecyl chains using non-contact AFM (nc-AFM), STM and molecular dynamic simulations (MD) at different temperatures. By comparing atomic-scale images of the assembly at 5 and 300 K we determined a giant positive thermal expansion of about 980 ± 110 × 1017  and sides (green) of the molecular skeleton. Each spoke axis is functionalized with a peripheral dodecyl side chain.The polyphenylene spoked wheel molecule (SW) 33. The spray-deposition was always performed on the Au(111) substrates kept at room temperature and followed by an annealing step at 450 K. As shown in large-scale STM measurements , we used an electrospray deposition (ESD) device attached to the UHV setup11, we usThe corresponding constant-height AFM image with a CO-terminated tip Fig. c furtherUpon spray-deposition of the spoked-wheel molecules in the sub-monolayer range, we observed different aggregates on the surface varying from single molecules up to extended assemblies  of about α = 14 × 10−6 K−1.A different molecule spacing of the networks is measured in Fig. Thermal evolution and mobility of a single SW molecule was numerically investigated using μs long all atom MD simulation on an Au(111) surface for different temperatures. Figure δchain at 5 K, 300 K, 420 K and 450 K, respectively. A larger δ corresponds to a larger mobility at a given temperature  is close to the neighbouring molecule (pink), that restricts its lateral fluctuation but also increases the intermolecular interactions. After the expansion event (>300 ns), alkyl chains possess enough space to adopt a more extended configuration. The relationship between chain mobility and lattice expansion is particularly visible between 400 and 700 ns in Fig. Additionally, the expansion occurs in distinguishable events as marked by dotted lines in Fig. Figure 1- a weak interaction with the surface (facilitating a rapid increase of their oscillations with temperature), 2- the soft in-plane bending stiffness of the chains (favouring this oscillation mode over other rigid vibrations) and 3- the two-dimensional constraint of the chain mobility imposed by the surface and excluding chain fluctuations out of the surface plane.The high lattice expansion may be understood as a conjunction of three properties of the dodecyl chains: Although the finite size of our simulations might affect the chain fluctuations and the intermolecular interaction, our simulations clearly support an abnormal thermal expansion coefficient for alkyl decorated molecules. Interestingly, small molecular assemblies obtained experimentally show a larger core-core distance  assemblies with and without alkyl chains (see Supplementary Note 4). In a recent work24, we studied the assembly of HBC molecules equipped with six dodecyl side chains (HBC-6C12H25) on Au(111) at 5 and 300 K by combined STM/AFM measurements . From the lattice measurements, we extract of a thermal expansion of this system of about α =  520 ± 60 × 10−6 K−1. While the same hexagonal ordering has been reported by Meissner et al. 34 from pristine HBC molecule , no lattice variation has been observed between 1.2 K and 300 K STM measurements, resulting in a thermal expansion of only α = 14 × 10−6 K−135.Besides unravelling the pivotal role of the alkyl chains into the giant thermal expansion of the SW assembly, we now discuss the generalization of this mechanism to interdigitated molecular systems at surfaces. Table 12H25 molecular assemblies experience a similar giant thermal expansion, that we attribute to the increased fluctuations of their interdigitated long alkyl chains governing the intermolecular assemblies. The cores of these precursors are quite different accounting for the slight difference in observed thermal expansion since the HBC lies flat on the surface24 while the SW core tends to protrude upon adsorption. In contrast to these interdigitated systems, supramolecular networks formed by pristine HBC molecules do only show a negligible expansion35. This observation again points towards the central role of the alkyl chains in the thermal expansion mechanism of interdigitated systems. Based on these observations, we believe that future works might address further the influence of the molecule cores as well as the effect of the chain lengths. We also anticipate that the structural phase transition might be also observed in such alkyl-based expansion mechanism24. This might enable tailoring the thermoresponse of such supramolecular systems.Both the SW and HBC-6Cα = 980 ± 110 × 10−6 K−1 value that can be explained by temperature-dependent fluctuations of alkyl side chains compared to the core of the molecules. The non-covalent intermolecular interaction governed by the alkyl chain interdigitation is thus the main factor responsible for the network expansion contrarily to the molecule core found to have a marginal role in the process. This mechanism is further generalised to two-dimensional interdigitated supramolecular systems at surfaces, in contrast to their pristine counterparts (without side chains) showing no thermal expansion. Such generalization might allow the rational design of new thermoresponsive 2D materials.We investigated the giant thermal expansion of a 2D supramolecular assembly composed of SW molecules on Au(111). Combining high-resolution STM/AFM images with MD calculations, we quantified a thermal expansion coefficient of + sputtering and annealing at 750 K. As result, atomically flat surfaces were obtained with large terraces separated by atomic steps.Au(111) single crystals (Mateck GmbH) were prepared in UHV conditions by several cycles of Ar−6 mbar. Typical applied voltage were 1.5–2.0 kV with adjustment sometimes necessary during the spray to maintain stable conditions. Depositions were performed for around 30 min and were followed by a slight annealing at approx. 450 K in order to remove any solvent contaminants. We reproduced such depositions in both UHV apparatus with identical parameters that always lead to molecular coverage well-below the monolayer and non-uniform molecular densities at the macroscopic scale. This allowed us to investigate both single molecules and extended assemblies on the same sample  samples kept at room temperature using a commercial system from MolecularSpray. The setup was connected to preparation chambers of both low and room temperature systems. The SW molecules were dissolved in a toluene and methanol mixture (ratio 5 to 1 in volume). During the spray deposition the pressure rose up to 1 × 1011 samplefst1 =  150 kHz and fnd2 = 1 MHz, oscillation amplitude 2–5 nm and 200–800 pm, respectively). Their preparation consisted in an annealing for 1 h at 400 K followed by a tip Ar+ sputtering for 90 s at 680 eV at an Ar+ pressure of p  = 3 × 10−6 bar. Base pressure of UHV system was maintained to 2 × 10−11 mbar during the measurements. The calibration of the (XY) AFM scanner at 300 K was checked by imaging the atomic surface lattice of a KBr(001) crystal.Room temperature nc-AFM measurements were performed with a home-built non-contact atomic force microscope with Nanonis RC5 electronics. PPP-NCL cantilevers (Nanosensors) were used as sensors . The STM experiments were conducted in the constant-current mode. The AFM experiments were performed in constant-height mode with a CO-terminated tips 21. The calibration of the (XY) scanner was checked by imaging the Au(111) surface with a CO-terminated tip.Low temperature STM/AFM measurements were realized with a low-temperature microscope (Omicron Nanotechnology GmbH) operated at 5 K with Nanonis RC5 electronics and based on a tuning fork sensor in the qPlus configuration 45—including its latest refinements (version 1.8 from 2017) available in AMBER18 36 that better reproduce the high-level ab initio and high quality experimental data of a variety of molecular properties36. At the intramolecular level, GAFF is able to accurately reproduce the structure and vibrational properties of a wide range of organic molecules 45 . Moreover, at the intermolecular level, GAFF has been routinely and successfully applied to study supramolecular polymers46, mechanically interlocked molecules 47, molecular assembly processes49 as well as other thermophysical properties that strongly depend on intermolecular interactions50 . As for the gold atoms, the CHARMM-METAL force-field 53 was applied, which simultaneously describe the intrinsic properties of gold, while retaining thermodynamical consistency with AMBER force field 53. This force field has been extensively tested by studying the adsorption of small organic molecules  against both density-functional-theory simulations as well as available experimental results53. Moreover, its suitability to describe the interaction with small organic molecules and their surface mobility has been recently validated through single molecule manipulation experiments54.An Au(111) surface composed by three atomic layers, where the positions of the atoms in the lowest layer were fixed during the MD runs using a harmonic restrain of 5 kcal molIn total six different microsecond long MD simulations, that can be grouped in two stages, were performed. The simulation details, notably the preparation stages prior to production runs, are provided in Supplementary Note Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5


The combination of scanning tunnelling microscopy (STM) and non-contact atomic force microscopy (nc-AFM) allows enhanced extraction and correlation of properties not readily available via a single imaging mode. We demonstrate this through the characterization and classification of several commonly found defects of the hydrogen-terminated silicon (100)-2 × 1 surface (H–Si(100)-2 × 1) by using six unique imaging modes. The H–Si surface was chosen as it provides a promising platform for the development of atom scale devices, with recent work showing their creation through precise desorption or placement of surface hydrogen atoms. While samples with relatively large areas of the H–Si surface are routinely created using an in situ methodology, surface defects are inevitably formed reducing the area available for patterning. By probing the surface using the different interactivity afforded by either hydrogen- or silicon-terminated tips, we are able to extract new insights regarding the atomic and electronic structure of these defects. This allows for the confirmation of literature assignments of several commonly found defects, as well as proposed classifications of previously unreported and unassigned defects. By combining insights from multiple imaging modes, better understanding of their successes and shortcomings in identifying defect structures and origins is achieved. With this, we take the first steps toward enabling the creation of superior H–Si surfaces through an improved understanding of surface defects, ultimately leading to more consistent and reliable fabrication of atom scale devices. Novel approaches to advance integrated circuitry beyond CMOS have focused on atom scale structures and their reliable fabrication . HydrogeNative silicon atoms at the unreconstructed (unterminated) (100) surface would, by argument of the crystal geometry, extend two unsatisfied bonds into vacuum. To minimize the surface energy, each silicon atom bonds with a neighbouring Si atom to create a dimer, thus reducing the number of dangling bonds (DBs) by half . Rows co2-Si). The 3 × 1 phase is a combination of the previous two, consisting of alternating 2 × 1 dimers and 1 × 1 dihydrides [The addition of hydrogen to surface silicon atoms saturates all available bonds  and threhydrides –22. Withhydrides –24  until an H2 molecule became trapped in the tip–sample junction. Here, we achieve STHM-like resolution by directly functionalizing the tip apex with a single hydrogen atom, picked up from the H–Si surface through the application of a voltage pulse, as reported in prior works [262 molecule dissociates on the apex, resulting in a singly H-functionalized tip [2 –34 and D2 [2  provided2 [2 . Rather or works –2636. Ouized tip . In Figuf(z)) taken above select positions on the surface (see f) at the same site [41A true measurement of the force interaction between the tip and sample can be visualized with frequency-shift maps generated by non-contact AFM . In our face see . The speame site –4143–45.e site 41–24 can bSimilar to how a change in tip functionalization can change the observed contrast  and 2) present an extra challenge to analyse, as they must be imaged with a larger tip–sample separation to prevent damaging tip contact. As a result, the magnitude of the frequency shift of the unperturbed surface is small compared to the strong defect signal. Despite this, examination of the cross-sections for the SiH3 and SiH2 respectively, resolves the position of the defect with respect to the lattice; the SiH3 originates above one side of a dimer, while the SiH2 is centred between the two atoms of a dimer.The two raised Si species in The neutral point defect in Lastly, a lone DB shows up as a localized feature of enhanced reactivity when compared to the surrounding H-terminated surface ,t. This We now focus on an in-depth analysis of the silicon vacancy defect introduced in We now return to a discussion of the neutral point defect, which we noted initially exists in a negatively charged, H-decorated state. I(V) and dI/dV spectroscopy of the neutral point defect can be seen in 2) is higher than would be expected for contaminant boron from commercial wafer processing [Furthermore, our assignment that the neutral point defect acts as a hydrogen trap during sample preparation is based on the similarity of the defect’s negative state to physisorbed hydrogen atoms on the surface -2 × 1 surface analysed using a combination of several STM and nc-AFM imaging modes, with reproducibly formed tip terminations of different reactivity. Through this analysis we are able to identify unique electronic and structural signatures associated with the defects of each imaging mode. By combining these results with a simple probe particle simulation of the STHM and AFM images, we were able to confirm the underlying mechanism of STHM, the classification of several surface defects, as well as more confidently identify the previously reported T2 defect as having character more consistent with a silicon vacancy. Finally, we examined the previously unreported neutral point defect, observing its transition from a negatively charged species by means of tip-induced liberation of an atomic hydrogen from the defect site. While our work presents a comprehensive understanding of the experimental nature of these defects, additional theoretical studies using more powerful techniques such as DFT is still needed to confirm the results presented here and provide a potential classification of the point defect. We also note that it is expected that other sample termination methods, Si wafers, and vacuum systems could potentially lead to additional defects not reported here. While this means this is not an exhaustive list, our analysis provides fresh insight into the nature of many commonly observed defects. Through this now enhanced understanding of the nature of the most common defects, we enable informed refinement of standard H–Si wafer preparation methods, leading to a more reliable platform for the creation of these devices.−11 Torr). STM tips were electrochemically etched from polycrystalline tungsten wire, resistively heated, and field-evaporated to clean and sharpen the apex using a field ion microscope (FIM) [f0 ≈ 28 kHz, Q-factor ≈ 16k–22k) [26Experiments were performed using an Omicron LT STM and an Omicron qPlus LT AFM –89 systepe (FIM) . AFM tip16k–22k) . The tip16k–22k) . In situ16k–22k) ,92. Brig16k–22k) –2636, pr19 atoms/cm3) Si(100). Samples were degassed at 600 °C overnight followed by multiple cycles of flash annealing at 1250 °C. The samples were then terminated with hydrogen by exposing them to molecular hydrogen (10−6 Torr) while the Si sample was held at 330 °C for 2 min. The molecular hydrogen was cracked from H2 gas using a tungsten filament held at 1600 °C [The samples used were highly arsenic-doped (≈1.5 × 10 1600 °C .f(z) spectroscopy changed between tip shaping events, but the general shape and behaviour for H- and Si-terminated tips (bright and dark contrast) remained consistent throughout multiple tips and tip terminations.Image and data acquisition was done using a Nanonis SPM controller and software, with the imaging parameters for each of the 6 SPM analysis modes described in the text. The height setpoint reference was taken as the tip–sample separation over a H–Si atom with an imaging bias of −1.8 V and a current setpoint of 50 pA. The exact magnitude of the ΔThe defect free H–Si ball and stick model was the same as used in , with deFile 1Referenced images and a supporting discussion of the probe particle simulations.


Flavoparmelia caperata that extends the physiological nature of symbiotic co-dependent metabolisms, mutually boosting energy conversion rates in both symbionts. Measurements of electron transport by oximetry show that photosynthetic O2 is consumed internally by fungal respiration. At low light intensity, very low levels of O2 are released, while photosynthetic electron transport from water oxidation is normal as shown by intrinsic chlorophyll variable fluorescence yield (period-4 oscillations in flash-induced Fv/Fm). The rate of algal O2 production increases following consecutive series of illumination periods, at low and with limited saturation at high light intensities, in contrast to light saturation in free-living algae. We attribute this effect to arise from the availability of more CO2 produced by fungal respiration of photosynthetically generated sugars. We conclude that the lichen symbionts are metabolically coupled by energy conversion through exchange of terminal electron donors and acceptors used in both photosynthesis and fungal respiration. Algal sugars and O2 are consumed by the fungal symbiont, while fungal delivered CO2 is consumed by the alga.Lichens are a symbiosis between a fungus and one or more photosynthetic microorganisms that enables the symbionts to thrive in places and conditions they could not compete independently. Exchanges of water and sugars between the symbionts are the established mechanisms that support lichen symbiosis. Herein, we present a new linkage between algal photosynthesis and fungal respiration in lichen The online version of this article (10.1007/s11120-019-00702-0) contains supplementary material, which is available to authorized users. However, the sources of O2 and CO2 gases and the mechanisms controlling their internal delivery and exchange have not been widely investigated and, until now, have been assumed to originate exclusively from environmental sources.Symbiotic relations have been vital throughout evolution to create new forms of life and support survival in challenging environments  and 86 to 1300% water content for optimal photosynthesis , which represents the flux of photosynthetic electron transport from water oxidation at the Photosystem II-Water Oxidation Complex (PSII-WOC). We also monitor PSII charge separation and water oxidation yield using intrinsic chlorophyll variable fluorescence yield (Fv/Fm) which is independent of possible O2 consumption. We aim to answer the following questions: How do algal O2 production and dark respiration rates change upon exposure to multiple light intensities, in aerobic and anaerobic conditions? How do internally produced O2, sugars and CO2 influence subsequent O2 production and consumption rates in response to prior illumination and dark periods?In this study, we investigate whether exchange of O2 and CO2 exchange between algal and fungal tissues. This discovery extends the earlier understanding of lichen symbiosis beyond exchange of photosynthetic sugars and water to encompass the gaseous products that literally power both types of energy production through fungal respiration  and algal photosynthesis .This study provides the first evidence for functional OFlavoparmelia caperata, a symbiosis between an ascomycete fungus and the green algal photobiont Trebouxia gelatinosa (Ahmadjian 2). This protocol achieves water-saturation as reported by Lange et al.  was used to measure O2 concentration released from samples immersed in a microcell . The Clark electrode has a thick membrane (~ 10 µm) and slow response that directly measures O2 concentration without significant consumption from the sample chamber over time. Light-induced O2 signals were produced using a LED light source .Two different oximetry methods were used to measure O2 concentration in the cuvette was recorded immediately, starting at aerobic conditions . Then lichen samples were alternatingly exposed to dark pre-conditions, subsequent light and subsequent dark conditions until all O2 was removed. After 100-min dark exposure to consume all O2 from the chamber, the same experiment was repeated at near-anaerobic initial conditions. Oxygen concentration was continuously measured as lichen disks were alternatingly exposed to dark and light conditions, the O2 data acquisition rate was 10 per second.Lichen disks, once inserted into the cuvette of the Clark electrode, were sealed from air and stirred by magnetic bar at 500 rpm. The O2 flux released from samples immersed in a thin-layer microcell  . This technique enables observation of O2 transients coupled to electron acceptors within PSII, PSI, and CO2 assimilation in the Calvin cycle. This behaves as a rate electrode which consumes O2 from the sample chamber and when the chamber is sealed, the small volume and large area allows anaerobic conditions to be established rapidly. The units for this electrode are in nA (current), which is directly proportional to the amount of O2 consumed per unit time by the electrode. The measured current is produced by O2 that is not consumed by the sample in the sealed chamber.A custom-built rate electrode comprised of a Pt–Ir alloy was used for measurement of O2 flux was measured from an intact lichen disk (4 mm diameter), exposed to continuous illumination from a red LED light source (655 nm) at two different light intensities of 70 and 800 μmol m−2 s−1 and in two different orientations (upper and bottom surface exposed to the O2 electrode). Each continuous illumination period lasted 90 s, after which the light source was turned off, while measurements continued for another 150 s. The initial (1st) illumination period was preceded by 30-min dark adaptation, after which 10 to 50 illumination periods were applied, separated by 10-min dark time between each illumination. The O2 data acquisition rate was 10 per second.Using this electrode, O2 yield was measured from individual light-saturating flashes (STFs), each 50 microseconds in duration and delivered at a frequency of 0.5 Hz in a train of flashes produced by the same LED (λ = 655 nm). The O2 current was integrated between flashes to obtain the yield. This classic method enables observation of period-4 oscillations in O2 yield known to be produced by all oxygenic phototrophs, including free-living algae  at a maximal flash intensity of 32,000 µmol m−2 s−1 (Ananyev and Dismukes A), is reduced to the semiquinone QA−. This closes the reaction center to further charge separation and increases the fluorescence emission yield from its dark-adapted minimum (Fo), arising from antenna chlorophyll emission, to its maximum emission (Fm), arising from both antenna and reaction center emission. The ratio Fv/Fm = (Fm − Fo)/Fm is directly proportional to the quantum yield of primary charge separation in PSII reaction centers . The FRR method of Chl fluorescence induction has been extensively applied in numerous studies of PSII in algae and in lichens  fluorometer utilizing a laser diode excitation source . The gross O2 production rate (μM h−1) is 490 before and 615 after light exposure, calculated as the difference between net O2 production and respiration rates before and after light exposure, respectively, assuming mitochondrial respiration rates continue at the same rate in light as in darkness before or after illumination. The linearity of regions D1 and D3 indicates that the respiration rates are independent of the varying O2 concentrations over these ranges. Finally, the respiration rate decreases exponentially below ~ 25 μM O2 concentration  enables determination of net O2 production and respiration rates, independent of externally available O2. The net O2 production rate is approximately constant for 10 min at 375 μM h−1, at which point the light was turned off  can be explained, in principle, by lower fungal respiration and/or higher gross O2 production rates. The respiration rate in the dark (D6) after illumination (L2), is − 240 μM h−1, about 11% higher compared to that under initial aerobic conditions (− 240 versus − 215 μM h−1), yet considerably lower than that under aerobic conditions after light exposure (− 240 versus − 340 μM h−1). The gross O2 production rate is approximately 615 μM h−1 (L2–D6), essentially identical to that under initial aerobic conditions (L1–D3). The gross photosynthetic O2 production rate equates to 20 μM g−1 dwt s−1 when normalized to dry weight. The kinetic response during transitions from dark-to-light and light-to-dark differs dramatically for aerobic versus anaerobic samples, being much sharper for the initially anaerobic sample. The respiration rate decreases exponentially below ~ 15 µM O2 . Respiration rates are typically higher after illumination than in aerobically dark-adapted samples . Gross O2 production rates (L1–D3 and L2–D6) vary as a result of biological variability of field samples as expected, however, they are very similar for initial aerobic versus anaerobic conditions (difference 0–13%). Results across biological samples illustrate that respiration rates increase following illumination and that net photosynthetic O2 production rates increase following respiration, while preserving an approximately constant gross production rate. This indicates that the two processes are metabolically linked and are capped at peak rates at high light intensity (800 μmol m−2 s−1).Table 2 fluxes was further investigated using the custom O2 rate electrode. This enables identification of transient changes in oxygen flux, the electrode responding rapidly to changes in O2 in the chamber because of its thinner membrane and tiny volume. Figure 2 rate measured by the electrode upon exposure of a lichen disk (4 mm diameter) to sub-saturating light intensity, 70 μmol m−2 s−1. A very small amount of O2 is detected directly after illumination starts, after which O2 rate gradually decreases over the next ~ 30 s followed by a linear increase until light is turned off. In the subsequent dark period, initially fast O2 consumption is followed by a more gradual decrease as the electrode continues to consume O2 in the dark. This results in a lower starting current for the second illumination trace and every subsequent trace .The effect of illumination on OChlorella vulgaris. O2 response upon exposure to light is very different from the lichen response: a steep increase in O2 is observed directly upon illumination, followed by a more gradual increase up to a peak value of ~ 5000 nA. There is no lag phase and the O2 response pattern is repeated identically for subsequent illumination traces, apart from an offset in starting levels as a result of O2 consumption by the electrode during intermediate dark times. When normalized to Chl content, the O2 evolution activity is ~ 500 μmol O2 (mg Chl)−1 h−1, typical of Chlorella algae , comparing different orientations of the lichen disk towards the electrode. We compare results for upper surface facing the electrode, where the algal layer in the lichen thallus is situated, versus the bottom surface facing the electrode, which is exclusively fungal material . While absolute values vary between biological samples, repeated experiments with samples from different lichen thalli show a consistent 1.5- to 3-fold increase in the slope of linear O2 evolution, comparing mean of 10 illuminations, 11–20 versus 1–10 .Figure Honegger . Traces −2 s−1 was applied to test the O2 response at light intensity that normally saturates photosynthesis in free-living algae. The O2 yield for 50 successive illumination traces is presented in Fig. 2 amplitude over the first 8 traces , after which the amplitudes gradually decrease, to a peak value of 4184 nA at the 50th trace. Averages over sets of 10 illumination traces in Fig. 2 rate both during illumination and subsequently in the dark become steeper between the first and second sets of illuminations (1–10 and 11–20), while both slopes gradually decrease on subsequent traces . Additionally, the O2 production rate saturates before each trace completes, i.e., is no longer linear, indicating that a lower rate is reached. At this higher light intensity, a transition period is observed after light is turned off, where O2 rates continue to increase gradually until reaching a peak, then fall as O2 consumption becomes dominant. The timing of this point shifts closer to the point where light is turned off, comparing illuminations 1–10 versus subsequent sets of illuminations.A higher light intensity of 800 μmol m2 production in lichen, we measured O2 yield in response to a train of single-turnover flashes (STF) from a lichen disk as well as from algal cells isolated from the same lichen disk. The lichen disk produced no detectable O2 over 50 flashes and no visible period-4 oscillations. . Simulations of the decay of the oscillations using VZAD, a standard WOC cycle model  actually occurs in intact lichens even though so little O2 is released at low light intensities, we measured period-4 oscillations of Chlorophyll variable fluorescence (Fv/Fm) in response to a train of STFs. To illustrate this, we show Fv/Fm response to 50 STFs from a lichen disk (7 mm diameter), in fully hydrated conditions  to the fungus to serve as electron donor for fungal respiration H) at the locations where dark respiration takes place (mitochondria). The observation of higher linear respiration rates by the fungus after each successive illumination and at higher light intensities indicates that the delivery of reductant to the terminal respiratory enzyme is increased, causing the observed increase in electron flux to O2. Under anaerobic conditions , the only source of O2 is that produced photosynthetically by the alga. This implies that increased fungal respiration rates following prior illumination, as observed in our experiments, confirm that both photosynthetic O2 (that we measure) and sugars (that are required to deliver reductant for respiration) are delivered by the alga to the fungus. Sources of NAD(P)H for respiration include catabolism of environmental carbohydrates and photosynthetic carbohydrates formed in the Calvin cycle (C3 to C6 sugars). The only source of exchangeable reductant that changes with illumination is photosynthetic carbohydrates produced by the alga.The observed increase in O2 and/or sugars produced by the algae are used by the fungus, as this is the only source available to support an increase in respiration (ambient conditions being kept the same). Since no evaporation occurs (the lichen is immersed in water), only diffusional transport drives the flow of algal products (sugar and O2) through the lichen thallus and to the fungus. As this transport is much slower for sugar than for O2, the mostly likely source of increased fungal respiration at the time-scale of these experiments is O2. We conclude that the algal–fungal symbiosis encompasses both fungal consumption of algal sugars and the photosynthetically produced O2. This is the first report that we are aware of showing O2 exchange between lichen symbionts and its metabolic role in boosting respiration rates.Both O2 consumption by the lichen is confirmed by our complementary oximetry and fluorometry (Fv/Fm) experiments . This is expected, since the WOC cycle is known to be irreversible and does not slow upon O2 partial pressures changing between zero and 20 bars  at external O2 , are all accounted for by linear electron flow from water to successive downstream electron acceptors  are depleted  or sink (decrease in O2 consumption) mechanism. An increase in photosynthetic O2 production rate (electron transport: H2O →→ NADP+ → CO2) can arise if the terminal electron acceptor concentration increases, owing to the greater amount of CO2 coming into the alga from fungal respiration. In turn, the increased fungal respiration originates from the increased Calvin-cycle sugars excreted by the alga to the fungus during successive pre-illumination cycles. Greater availability of photosynthetic O2 within the lichen is not expected to increase fungal respiration alone (without co-release of sugar), for O2 concentrations above saturation.The observed increase in slope of the linear COon Figs. , 4 can b2 consumption (respiration or binding) by the fungus can increase the slope of the linear CO2-dependent phase of photosynthetic O2 evolution with successive illuminations. However, O2 consumption rates in the dark increase upon subsequent illuminations . Only after multiple illumination cycles, beyond 20–30 illumination cycles at high light intensity, does the net photosynthetic O2 evolution rate decreases which we attribute to consumption of fungal-respired CO2  that is induced by internal CO2 production and delivery from the mycobiont to the photobiont in order to boost O2 production under low internal CO2 conditions. Its simultaneous O2 and CO2 exchange between symbionts distinguishes it from CCMs that exist in free-living cyanobacteria and algae, where internal CO2 stores are filled during illumination and subsequently released in the dark  and suggest that “it is probably less effective than that which operates in cyanobacterial lichens.” By contrast, our results using oximetry and fluorometry indicate that this interpretation—a single organismal source mechanism to account for the much smaller pool of CO2 released in darkness after illumination in green algal lichens—is insufficient and actually arises from the much greater capacity of the green algal photobiont to consume fungal-generated CO2 by an inter-species exchange mechanism.The CO2 after light is turned off that occurs especially at high light intensity  to CO2 occurs at light intensities that would completely light-saturate O2 evolution in free-living algal cells. We conclude that the algal–fungal symbiosis of lichens is mutually beneficial to the metabolism of both organisms at the fundamental level of electron transport in both photosynthesis and dark respiration, including electron transport for both metabolisms . The algal and fungal energy metabolisms are mutually linked, creating a two-way turbo-charged symbiosis.In this work, we investigated oxygen production and respiration in lichen Supplementary material 1 (DOCX 2146 kb)Below is the link to the electronic supplementary material.


Vitis vinifera) teinturier cultivars are characterized by their typical reddish leaves and red-fleshed berries due to ectopic anthocyanin formation. Wines of these varieties have economic importance as they can be used for blending to enhance the color of red wines. The unique and heritable mutation has been known for a long time but the underlying genetic mechanism still is not yet understood. Here we describe the association of the red-fleshed berry phenotype with a 408 bp repetitive DNA element in the promoter of the VvmybA1 gene . Three different clones of ‘Teinturier’ were discovered with two, three and five allelic GCE repeats . All three clones are periclinal chimeras; these clones share the same L1 layer, but have distinct L2 layers with different quantities of GCE repeats. Quantitative real time PCR and HPLC analysis of leaf and berry samples showed that the GCE repeat number strongly correlates with an increase of the expression of VvmybA1 itself and the VvUFGT gene regulated by it and the anthocyanin content. A model is proposed based on autoregulation of VvmybA1t to explain the red phenotype which is similar to that of red-fleshed apples. This study presents results about the generation and modes of action of three MybA1t alleles responsible for the red-fleshed berry phenotype of teinturier grapevines.Grapevine ( Vitis vinifera L.), the important fruit traits with a broad spectrum ranging from green/yellow to blue/black are linked with anthocyanin-based berry color formation [O-glucoside, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, peonidin-3-O-glucoside and malvidin-3-O-glucoside) and their acylated derivatives [Anthocyanins (from Greek “anthos” = flower and “kyanos” = blue) represent a ubiquitous class of metabolites in nature . They beormation . The theivatives ,6.F3′H and F3′5′H, respectively [O-glucosyltransferase) resulting in the color-intensive pigments [O-methyltransferases, which methylate hydroxyl groups at the 3′ and 5′ positions of the B ring [The anthocyanin biosynthesis pathway shares with all flavonoids the same upstream pathway that branches at the level of flavanones ,8. The tectively ,10. All pigments . Furthere B ring  and acyle B ring . Due to e B ring .VvUFGT [VvUFGT is controlled by a single locus on chromosome 2, also called berry color locus, at the physical position of about 14.2 Mb according to the reference genome sequence [F3′H and F3′5′H expression [VvmybA transcription factor genes of the R2R3-type with the key genes VvmybA1 and VvmybA2 active during ripening in the berry skin [VvmybA genes, white varieties show only non-functional VvmybA genes at the berry color locus. In case of the non-functional VvmybA1 allele, called VvmybA1a or MybA1a, the retrotransposon Gret1 upstream of the coding sequence hampers the expression, whereas the functionality of VvmybA2 is inactivated by two non-conservative mutations, an amino acid substitution and a frame shift, resulting in an early stop codon and a truncated protein. The functional VvmybA1 gene instead, called VvmybA1c or MybA1c, completely lacks Gret1 and is considered dominant over VvmybA1a [In grapevine, the relative abundance of anthocyanins and total quantities mainly are controlled genetically, developmentally regulated and variety-specific, but environmental factors such as light, temperature and water availability also influence the phenotype. Excessively high temperatures and too much light can have negative effects on berry color, whereas water deficit promotes berry anthocyanin content ,16,17. AVvUFGT . On genesequence ,20. Furtpression . The colrry skin ,22. In cVvmybA1a .Anthocyanins are synthesized typically in the skin but not in the pulp of grapevine berries starting with the onset of ripening (veraison = softening of the berry). In contrast, the grapevine variety ‘Teinturier (du Cher)’ and its derivatives  deviate from this natural situation by accumulating variable anthocyanin contents in the leaves, the berry flesh and the berry skin. These varieties show in general a more or less intense red coloration of the foliage and stems during the growing season and dark red colored autumn leaves . The skiBerry color mutations are easily detectible in the vineyard by simple visual inspection; many clones with different colors than their parent variety have been selected since the rise of viticulture . In thisVvmybA1 we analyzed the coding sequence and the adjacent promoter region of a large set of teinturier varieties showing a range of berry and vine phenotypes. We compared red fleshed clones and varieties of the ‘Teinturier’ lineage to wild type varieties by examining the sequences of the VvmybA1 region, expression data of anthocyanin biosynthesis genes, and anthocyanin content and composition of berry and leaf tissues. Based on these results, we propose a model that links the unique mutation on a genetic level with the distinct red berry flesh and other phenotypes of teinturier varieties.The main goal of this study was to characterize the specific mutation of the grapevine teinturier varieties of the ‘Teinturier’ lineages differing in color intensity of their red-fleshed phenotype. Due to the altered organ-specific expression of ‘Teinturier 3x’ and ‘Teinturier 5x’ (‘Rubintraube’) were obtained as dormant wood cuttings from the Bundessortenamt, Prüfstelle Hassloch, Germany and from the Staatliche Lehr- und Versuchsanstalt für Wein- und Obstbau, Weinsberg, Germany, respectively. All other grapevine accessions used were maintained in the vineyards of the germplasm repository at JKI Geilweilerhof, Siebeldingen, Germany. Potted plants were produced after leaf fall from woody cuttings during the winter season of 2015 and cultivated in the greenhouse. Berry samples were taken from field-grown vines; ten ripe berries of three sun-exposed bunches were collected at three developmental stages ). Berry skin and flesh were immediately separated and the seeds were removed. Four to six leaves were randomly collected from six greenhouse-grown vines that were eight months old.http://www.vivc.de/). Six additional SSR markers were used to determine the haplotype structure around the berry color locus on chromosome 2  the primers “a” and “c” were taken from Kobayashi et al. [VvmybA1c allele, primers were located outside of the open reading frame. MybA1_prom_fw (5′-TCAGTGAGGGTAACAAAGTC-3′) and MybA1_prom_seq_fw (5′-TAAAGCCTATAATATTCCCAC-3′) were positioned in the 5′ flanking region 808 bp and 1237 bp upstream of the start codon and used for repeat number validation and sequencing, respectively. MybA1_term_rev (5′-CAAGCTAGAAAGAGAGATGTGT-3′) was used for both purposes and positioned in the 3′ flanking region 494 bp downstream of the stop codon. All primers were purchased from Metabion . PCR for the repeat number validation was performed in a final reaction volume of 25 µL containing 20–30 ng genomic DNA, 0.3 mM dNTPs, 1 x KAPA HiFi Buffer, 0.3 mM of each primer and 0.5 U polymerase . A Mastercycler® gradient  was used for amplification with the following touchdown cycle program: initial activation at 95 °C for 5 min; followed by 10 cycles of 98 °C for 20 s, 65 °C for 15 s (−0.5 °C each cycle temperature increment) and 72 °C for 120 s; followed by 20 cycles of 98 °C for 20 s, 60 °C for 15 s and 72 °C for 120 s; then a final extension at 72 °C for 5 min. To better determine the size of the amplified 5′ flanking regions of VvmybA1 from ‘Teinturier’ clones, PCR products were purified with the Kit NucleoSpin® Gel and PCR Clean-up  and restriction digested with EcoRI . DNA fragments were separated by electrophoresis on a 1% agarose gel . For sequencing of the VvmybA1 alleles, a Touchdown-PCR was conducted with the conditions described above. In the cycle program, the annealing temperature was 61 °C and the elongation time was increased to 150 s due to the longer PCR product. PCR products were purified and Sanger sequenced by GATC Biotech AG .Genomic DNA was isolated for a cell layer-specific PCR analysis as described in Vezzulli et al. . We extri et al. . To ensug rpm and 50 µL of the supernatants were analyzed for anthocyanins on an Agilent 1100/1200 series HPLC system with a diode-array detector . Separation was conducted on a LiChrospher 100 RP 18 (5 µm) column in LiChroCart 250-4 protected by a LiChroCart 4 mm RP 18 guard column  as described in the OIV-MA-AS315-11 method [v/v/v) and solvent B  was applied at a flow rate of 0.4 mL/min and a isothermal column temperature of 20 °C as follows: 94–70% A and 6–30% B from 0 to 15 min, 70–50% A and 30–50% B from 15 to 30 min, 50–40% A and 50–60% B from 30 to 35 min, 40–94% A and 60–6% B from 35 to 41 min and a final step with 94% A and 6% B for re-equilibration of the column from 41 to 50 min. The detection wavelength for anthocyanins was set at 520 nm and the quantification was carried out by peak area measurements. The anthocyanin amount was expressed by using malvidin-3-O-glucoside as standard  for a calibration curve (R2 = 0.99) as described in [Anthocyanins were extracted from ground tissues (0.08–0.15 g) in 10 mL of an ethanol:water:hydrochloride acid solution (70:29:1) via overnight shaking at 100 rpm in the dark as previously detailed . Extract1 method . A gradiribed in ,43. ® MinElute® Cleanup Kit (Qiagen) following the supplier’s protocol. For cDNA synthesis, 100–1000 ng of total RNA was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit  following the manufacturer’s instructions. Quantitative real-time PCR for expression analysis was performed on an ABI 7500 Fast PCR System  using SYBR®-Green Real Time PCR Master Mix  with 10 ng cDNA and 250 nM primer in each reaction with the following thermocycling conditions: initial activation at 95 °C for 10 min; followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s; then a melting curve analysis according to the manufacturer’s instructions. Relative quantification was performed by means of the 2−ΔΔCt method [VvmybA1 was MybA_RT_fw (5′-GAGTTTGCATTAGACGAGGTT-3′) and MybA1_RT_rev (5′-CTTTTTGAAGTGGTGACT-3′). For VvF3′H and VvF3′5′H primer sequences were derived from [VvUFGT were taken from [Total RNA was extracted according to the protocol described by  with twot method  using SDt method . Primer t method , for GAPken from .http://ensembl.gramene.org/Vitis_vinifera/Info/Index). The same software was used to assemble the sequence reads of the different VvmybA1 alleles. The promoter sequence of the VvmybA1 allele was screened for putative MYB protein binding domains with the databases PlantCARE [V. vinifera varieties, each genotyped with 20 SSR markers, was used to estimate allele frequencies within the V. vinifera population for a likelihood ratio test. The Student’s t-test and Tukey’s HSD test of the R-software environment were used to determine the statistical significant differences of anthocyanin and gene expression data [Oligonucleotides were designed using CLC Main Workbench 7  based on the PN40024 grapevine reference genome ,50 provilantCARE  and PLAClantCARE . For estlantCARE . The SSRlantCARE  consistiion data .VvmybA1 at the color locus. For a detailed study of the molecular basis of this mutation, a set of 21 different grapevine accessions were chosen consisting of 15 ‘Teinturier’ clones, five varieties descended from ‘Teinturier’ through crossing or hybridization and ‘Pinot Noir’ as negative control. To verify the relatedness among the accessions, they were genotyped with nine genome-wide SSR markers and with six markers covering a region of about 327 kb around the berry color locus on chromosome 2 . The observation of putative three alleles in genomic DNA of leaf tissue DNA consisting of L1 and L2 cell layers in ‘Teinturier’ resembled previous studies on grapevine cultivars ‘Pinot Meunier’ and ‘Pinot Gris’, proven to be periclinal chimeras [For the direct examination of the  samples A. The 1.a allele  and the c allele . Intereschimeras ,58.MybA1c-related 2.2 kb band was missing in all ‘Teinturier’ clones, whereas only the white MybA1a allele and one unknown allele variant could be amplified . The PCR analysis showed that in the L2-specific DNA samples the mplified A. These VvmybA1 allele variants of the ‘Teinturier’ clones were sequenced to verify their identity and to search for the reason for the length polymorphisms. Sequence comparison of these new allele variants with the VvmybA1c allele of ‘Pinot Noir’ and that of the L1 layer of ‘Teinturier’ showed perfect identity, thereby supporting the full-sibling kinship of ‘Pinot Noir’ and ‘Teinturier’ as mentioned before. An alignment of the MybA1c allele of ‘Pinot Noir’ and the unknown allele sequence of ‘Teinturier’ is shown in EcoRI and separated on an agarose gel for better estimating the size of fragments. The unique EcoRI site was located 22 bp downstream of the repeat site and cleaved the PCR products in a constant 3′ fragment (1768 bp) and a variable 5′ fragment of 493 bp (wildtype), 901 bp (two repeats), 1309 bp (three repeats) and 2125 bp (five repeats)  indicating the origin and the number of repeats.The three new putative repeats) . The thrMybA1t2 allele, three MybA1t3 and two MybA1t5  in the 5′ upstream region of the VvmybA1t alleles is associated with the colored berry flesh phenotype in teinturier varieties.Ten of the analyzed ‘Teinturier’ accessions possessed the  MybA1t5 . The speVvmybA1t promoter of the latter accession suggested that the number of repeats affects the anthocyanin content. To test this hypothesis, the near-isogenic ‘Teinturier’ clones differing only in their GCE number  were cultivated in the greenhouse. The visual inspection of the eight-month-old plants showed enhanced leaf coloration with increased GCE number  or the variety ,24. Therrier 5x’ . The expVvF3′5′H C,D. The MybA1t allele and additionally a non-functional MybA1a allele to avoid possible side effects of a second functional MybA1c allele. The following two varieties per MybA1t allele type were chosen: (i) MybA1t2: ‘Teinturier 2x’ and ‘Dunkelfelder’; (ii) MybA1t3: ‘Kolor’ and ‘Deckrot’; (iii) MybA1t5: ‘Cabernet Mitos’ and ‘Palas’. The full pedigree tree of these varieties including the putative kinship of the ancestor ‘Teinturier’ is displayed in Fruit anthocyanin content is much more important in grape growing and winemaking than leaf anthocyanin content. ‘Teinturier’ descendants growing in the field and bearing fruit were phenotypically and genetically checked to be true teinturier grapevines with red-fleshed berries. To be genetically as close as possible to the original ‘Teinturier’, all varieties chosen were direct offspring of ‘Teinturier’ (F1 generation). They all were checked to carry, like the parent ‘Teinturier’, one MybA1t increased , teinturier grapes often begin anthocyanin accumulation before veraison in the pea-sized stage A. At thiromoters B. For VvVvmybA1 transcription factor gene leads to the red berry flesh and foliage phenotype in red-fleshed varieties descended from ‘Teinturier’. In previous research, the 408 bp indel was associated with the teinturier phenotype, but the repeating character of this sequence motif was not identified [VvmybA1 gene of a larger set of ‘Teinturier’ grapevine accessions revealed a 408 bp repeated element (GCE) in the promoter region with variations of two, three or five copies  in the vicinity of the start codon. With the exception of the GCE duplication, the coding sequence itself and the adjacent up and downstream sequences , depending on whether anthocyanins are present in the berry skin during ripening or not. As berry color mutations easily can be recognized by visual inspection, many sports or clones with berry colors ranging from green/yellow to blue/black have been selected since the rise of viticulture, increasing today’s variety spectrum. The molecular origin for many mutants has been elucidated and is associated with the berry color locus on chromosome 2 ,59. The entified ; this isMybA1t gene is very difficult in genotypes with functional (red) MybA1c and (red) MybA1t haplophases due to identical and competitive primer binding sites and the discrimination towards shorter PCR products. However, after focusing the PCR analysis on L2-specific root tissue  MybA1a and functional (red) MybA1t) and resolving the experimental difficulties we identified the repetitive GCE as clearly associated with the red berry pulp and foliage of the original variety ‘Teinturier’ as well as in ‘Teinturier’ offspring. The differences in the coloration of leaves and young berries (prior to veraison) can already visually be observed in teinturiers  already exist in not yet analyzed ‘Teinturier’ individuals. It can be expected that allele variants with more than five GCEs would lead to even higher anthocyanin color concentrations than already observed in the MybA1t5 varieties if there are no negative selective constraints, such as deleterious effects of very high anthocyanin content in leaves. Most repetitive sequences can be classified as orientated short tandem repeats such as SSRs (simple sequence repeats) or longer transposable elements interspersed in the genome . In geneVvmybA1 clearly increased and correlated with the number of the repetitive elements in the promoter , PetuniMYBCORE, ), Solanu and Arang-site, ). Althoue et al.  who assupression . An enhaMybA1a and a functional MybA1c allele and should display the original genetic constitution regarding berry color. The inner cell layer L2 has a non-functional MybA1a allele and one of the three MybA1t alleles, respectively. Since the complete sequence of MybA1t from the L2 cell layer, with the exception of the repeats, was identical to the MybA1c allele of the L1 layer, the new teinturier alleles arose most probably through mutation of the original MybA1c allele of the L2 layer resulting in a periclinal chimera. As gametes arise from the L2 lineage in dicots [Due to the centuries-long vegetative propagation of old cultivars in viticulture, the level of somatic variation within varieties descending from a single seedling is high . Somaticn dicots , the varn dicots . In the MybA1c allele. ‘Teinturier’ alleles show no additional differences when compared to the ‘Pinot Noir’ MybA1c allele (p-value of 0.0096, we could conclude that ‘Teinturier’ and ‘Pinot Noir’ are most likely full-siblings sharing the parents ‘Savagnin Blanc’ and an unknown variety with black berries that is the source of the functional color allele.Before ‘Teinturier’ mutated to the red-fleshed phenotype, the variety should have been a typical red wine cultivar with a functional c allele  and the c allele —these vaVvmybA1 gene leads to a higher expression of the VvmybA1 gene itself, downstream regulated genes of the anthocyanin biosynthetic pathway and finally the anthocyanin content. Variations of this GCE mutation with two, three and five repeats were found in different ‘Teinturier’ accessions and derived teinturier varieties, which lead to copy number-dependent stronger effects on gene expression and ectopic anthocyanin formation. At the same time, we showed that cultivar ‘Teinturier’” is a periclinal chimera; the original somatic mutation occurred in the L2 layer.We characterized a genetic mutation and its association with the typical phenotype of red berry flesh and foliage of the well-known teinturier grapevine varieties. A 408 bp insertion in the promoter of the cis-acting binding sites for transcription factors is modified. In addition to the mutation and associated phenotype of ‘Teinturier’, we described an example of how variability can further evolve at the mutation site due to putative recombination mechanisms based on microhomology. The three ‘Teinturier’ clones with increasing GCE number represent near-isogenic lines that are ideal for further studies to shed light on the quantitative control of anthocyanin biosynthesis. The results highlight the importance of somatic mutations for the vegetatively propagated and perennial grapevine. Grapevine shares molecular mechanisms with some red-fleshed mutants of other fruit crops. Somatic mutations increase genetic diversity and can affect important agronomic traits. The case of ‘Teinturier’ shows that in addition to mutations in the coding sequence of a gene, altered promoters can drive even greater phenotypic diversity, especially if the number of presumable