SUB1 has photoreceptor dependent and independent functions in sexual development and secondary metabolism in Trichoderma reesei.

Light dependent processes are involved in the regulation of growth, development and enzyme production in Trichoderma reesei. The photoreceptors BLR1, BLR2 and ENV1 exert crucial functions in these processes. We analyzed the involvement of the transcription factor SUB1 in sexual development as well as secondary metabolism and its position in the light signaling cascade. SUB1 influences growth and in contrast to its homologue in N. crassa, SUB1 is not essential for fruiting body formation and male fertility in T. reesei, but required for female fertility. Accordingly, SUB1 is involved in the regulation of the pheromone system of T. reesei. Female sterility of mutants lacking env1 is rescued in triple mutants of blr1, blr2 and env1, but not in double mutants of these genes. Confrontation of strains lacking sub1 results in growth arrest prior to contact of the potential mating partners. This effect is at least in part due to altered secondary metabolite production. Additionally, together with BLR1 and BLR2, SUB1 is essential for spore pigmentation and transcription of pks4, and secondary metabolism is regulated by SUB1 in a light- and nutrient dependent manner. Our results hence indicate rewiring of several pathways targeted by SUB1 in T. reesei.

Omics Analyses of Trichoderma reesei CBS999.97 and QM6a Indicate the Relevance of Female Fertility to Carbohydrate-Active Enzyme and Transporter Levels.

The filamentous fungus Trichoderma reesei is found predominantly in the tropics but also in more temperate regions, such as Europe, and is widely known as a producer of large amounts of plant cell wall-degrading enzymes. We sequenced the genome of the sexually competent isolate CBS999.97, which is phenotypically different from the female sterile strain QM6a but can cross sexually with QM6a. Transcriptome data for growth on cellulose showed that entire carbohydrate-active enzyme (CAZyme) families are consistently differentially regulated between these strains. We evaluated backcrossed strains of both mating types, which acquired female fertility from CBS999.97 but maintained a mostly QM6a genetic background, and we could thereby distinguish between the effects of strain background and female fertility or mating type. We found clear regulatory differences associated with female fertility and female sterility, including regulation of CAZyme and transporter genes. Analysis of carbon source utilization, transcriptomes, and secondary metabolites in these strains revealed that only a few changes in gene regulation are consistently correlated with different mating types. Different strain backgrounds (QM6a versus CBS999.97) resulted in the most significant alterations in the transcriptomes and in carbon source utilization, with decreased growth of CBS999.97 on several amino acids (for example proline or alanine), which further correlated with the downregulation of genes involved in the respective pathways. In combination, our findings support a role of fertility-associated processes in physiology and gene regulation and are of high relevance for the use of sexual crossing in combining the characteristics of two compatible strains or quantitative trait locus (QTL) analysis.IMPORTANCETrichoderma reesei is a filamentous fungus with a high potential for secretion of plant cell wall-degrading enzymes. We sequenced the genome of the fully fertile field isolate CBS999.97 and analyzed its gene regulation characteristics in comparison with the commonly used laboratory wild-type strain QM6a, which is not female fertile. Additionally, we also evaluated fully fertile strains with genotypes very close to that of QM6a in order to distinguish between strain-specific and fertility-specific characteristics. We found that QM6a and CBS999.97 clearly differ in their growth patterns on different carbon sources, CAZyme gene regulation, and secondary metabolism. Importantly, we found altered regulation of 90 genes associated with female fertility, including CAZyme genes and transporter genes, but only minor mating type-dependent differences. Hence, when using sexual crossing in research and for strain improvement, it is important to consider female fertile and female sterile strains for comparison with QM6a and to achieve optimal performance.

Overview of Methods for the Direct Molar Mass Determination of Cellulose.

The purpose of this article is to provide the reader with an overview of the methods used to determine the molecular weights of cellulose. Methods that employ direct dissolution of the cellulose polymer are described; hence methods for investigating the molecular weight of cellulose in derivatized states, such as ethers or esters, only form a minor part of this review. Many of the methods described are primarily of historical interest since they have no use in modern cellulose chemistry. However, older methods, such as osmometry or ultracentrifuge experiments, were the first analytical methods used in polymer chemistry and continue to serve as sources of fundamental information (such as the cellulose structure in solution). The first part of the paper reviews methods, either absolute or relative, for the estimation of average molecular weights. Regardless of an absolute or relative approach, the outcome is a molecular weight average (MWA). In the final section, coupling methods are described. The primary benefit of performing a pre-separation step on the molecules is the discovery of the molecular weight distribution (MWD). Here, size exclusion chromatography (SEC) is unquestionably the most powerful and most commonly-applied method in modern laboratories and industrial settings.

Self-organising maps for the exploration and classification of thin-layer chromatograms.

Thin-layer chromatography (TLC) allows the swift analysis of larger sample sets in almost any laboratory. The obtained chromatograms are patterns of coloured zones that are conveniently evaluated and classified by visual inspection. This manual approach reaches its limit when several dozens or a few hundred samples need to be evaluated. Methods to classify TLCs automatically and objectively have been explored but without a definitive conclusion; established methods, such as principal component analysis, suffer from the variability of the data, while contemporary omics methods were constructed for the analysis of large numbers of highly resolved analyses. Self-organizing maps (SOMs) are an algorithm for unsupervised learning that reduces higher dimensional datasets to a two-dimensional map, locating similar samples close to each other. It tolerates small variations between samples of the same type. We investigated the capability of SOMs for the evaluation of TLCs with two sample sets. With the first one (495 analyses of essential oils), it was confirmed that SOMs arrange the same type of sample in a common region. The obtained multi-class maps were used to classify a test set and to explore the causes for the few misclassifications (<3%). With the second test set (50 extracts of experimental wheats), the effects of a greater variability within substance classes was explored. With SOMs, it was possible to single out the exceptional samples that warranted a more detailed investigation. In addition, the SOM quality control index method was tested. It proved to be considerably stricter than the classification with a SOM of all samples. When this method was unable to classify a sample correctly, it would flag the sample for inspection, as it gave either multiple assignments or none at all. The combination of SOMs and TLC - two accessible analytical tools - can be most useful for the unsupervised classification of samples by TLC, and to identify samples that stand out from a set and are therefore worth the investment into additional analyses with more complex or expensive methods.

Antioxidant properties and qualitative analysis of phenolic constituents in Ephedra spp. by HPTLC together with injection port derivatization GC-MS.

Ephedra herb extracts are being extensively investigated in terms of their antioxidative, antimicrobial and antiproliferative properties, with phenolic components being the general carriers of these bioactivities. Here we describe a comprehensive set of analytical methods employed to determine and characterize both the antioxidative activity and the qualitative profile of phenolic acids and flavonoids present in several Ephedra species of different geographical origin. Spectrophotometric methods were used to determine the total phenolic content, total flavonoid content and antioxidative activity. Multi-development HPTLC enabled chemical fingerprinting which can be used for species differentiation. Individual spots of the thin-layer chromatogram were subjected to GC-MS with injection port derivatization for identification, which was based on both the detected mass spectra and recorded retention indices. The results were compared and complemented with GC-MS using offline derivatization.

Phenotypic and genotypic diversity among strains of Aureobasidium pullulans in comparison with related species.

Intra-specific diversity of 200 Aureobasidium pullulans strains isolated from different sources and their relatives Kabatiella lini CBS 125.21 T and Hormonema prunorum CBS 933.72 T were studied by assessment of macromorphological, and physiological tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis technique (SDS-PAGE) of whole-cell proteins as well as enterobacterial repetitive intergenic consensus (ERIC)-, repetitive extragenic palindromic (REP)- and BOX-PCR techniques (collectively known as rep-PCR). Rep-PCR is an efficient procedure for discrimination of A. pullulans in terms of simplicity and rapidity. RFLP-PCR technique was applied for the identification of A. pullulans isolates and distinction from related species. This technique was insufficient for investigation of intra-specific diversity. The tested strains of A. pullulans could be divided into two groups based on their macromorphological, protein patterns obtained after SDS-PAGE as well as rep-PCR patterns. The first group of strains shared similar characteristics and was very different from the second one, designated as "complex group", consisting of strains with very little similarities within the group. Phenetic analysis of ERIC banding patterns failed to group the isolates on the basis of their substrate or geographical origin. Using 18S rDNA gene sequence analysis of selected isolates, three strains: HoHe3 km, A. pullulans DSM 62074 and H. prunorum CBS 933.72 T were distinguished from all other analysed members of genera Aureobasidium and Kabatiella.

Genetic diversity among isolates of Paenibacillus larvae from Austria.

Genetic diversity of 214 Paenibacillus larvae strains from Austria was studied. Genotyping of isolates was performed by polymerase chain reaction (PCR) with primers corresponding to enterobacterial repetitive intergenic consensus (ERIC), BOX repetitive and extragenic palindromic (REP) elements (collectively known as rep-PCR) using ERIC primers, BOX A1R and MBO REP1 primers. Using ERIC-PCR technique two genotypes could be differentiated (ERIC I and II), whereas using combined typing by BOX- and REP-PCR, five different genotypes were detected (ab, aB, Ab, AB and alphab). Genotypes aB and alphab are new and have not been reported in other studies using the same techniques.

Resource-Saving Production of Dialdehyde Cellulose: Optimization of the Process at High Pulp Consistency.

Oxidation of cellulose with periodate under aqueous conditions yields dialdehyde cellulose, a promising functional cellulose derivative. The main obstacles for this oxidation have been its slow kinetics and the dilute reaction conditions, requiring considerable amounts of water and energy. In this study, these drawbacks are overcome by conducting the oxidation at high cellulosic pulp consistency with a cellulose/water weight ratio of 1:4. The oxidizer, cellulose, and water are efficiently mixed in a ball mill. Oxidation occurs mostly in the subsequent step, during the resting time (no further milling/mixing is required). The reaction and resource efficiency of the process are optimized by experimental design and a maximum aldehyde content of 8 mmol g-1 is obtained with a periodate/cellulose molar ratio of 1.25, a milling time of 2 min, and a resting time of 8 h. The developed method allows fine tuning of the oxidation level and is a key step towards the sustainable periodate oxidation of cellulose also on larger scale.

Profiling and quantification of grain anthocyanins in purple pericarp × blue aleurone wheat crosses by high-performance thin-layer chromatography and densitometry.

BACKGROUND: Anthocyanins are abundant secondary metabolites responsible for most blue to blue-black, and red to purple colors of various plant organs. In wheat grains, anthocyanins are accumulated in the pericarp and/or aleurone layer. Anthocyanin pigmented wheat grains can be processed into functional foods with potential health benefits due to the antioxidant properties of the anthocyanins. The grain anthocyanin content can be increased by pyramidizing the different genes responsible for the accumulation of anthocyanins in the different grain layers. Our objective was to develop a high-performance thin-layer chromatography (HPTLC) method that allows the determination of both the anthocyanin profile and the total pigment concentration. Thereby, selection of breeding lines with significantly higher grain anthocyanin content from purple pericarp × blue aleurone wheat crosses should become more efficient than selection based on only visual scoring of grain color and the unspecific determination of anthocyanin concentration by UV/Vis spectroscopy. RESULTS: A wide variability in the grain anthocyanin content was observed in breeding lines and check varieties. The highest concentration of anthocyanins was observed in deep purple (i.e. combination of the purple pericarp and blue aleurone genetics) grained breeding lines, followed by blue aleurone and purple pericarp genotypes. Determination of the total anthocyanin content was included into the chromatographic analysis, rendering an additional photometric analysis unnecessary. Ten target zones were identified in anthocyanin pigmented wheat grains; four of these zones were typically for blue aleurone types, five for purple pericarp types, and one (i.e. kuromanin glucoside) was characteristic for both. Chemometrics applied to the anthocyanin profile recorded by scanning densitometry revealed that peak heights and peak areas are highly correlated and that seven out of the ten target zones were responsible for about 90% of the total variation in the germplasm. Multivariate analysis of these seven target zones allowed not only a separation of the genetic material into purple, blue and deep purple grained genotypes, but also the identification of genotypes with a specific anthocyanin pattern. Thereby, the original classification by visual scoring was overruled in about one-third of the breeding lines. CONCLUSIONS: The presented HPTLC method with à côté calibration allowed the profiling of the pigments and quantification of wheat grain anthocyanin content in a single analysis, replacing UV/Vis spectroscopy with subsequent HPLC analysis. Moreover, no sample preparation apart from extraction and filtration is required, and more than 15 samples can be evaluated in one analysis run, corresponding to several dozens of samples per day. Hence, the method fulfills the requirements for screening methods in early generations of a plant breeding program such as high-throughput, small sample size, high repeatability, fast determination, and reasonable costs per sample. Combined with multivariate statistical analysis, the anthocyanin pattern allowed the validation of the genetic background in the offspring of purple × blue wheat crosses and, therefore, the efficient selection of genotypes exhibiting both the cyanidin and delphinidin aglycon.

À côté calibration - Making optimal use of time and space in quantitative high performance thin layer chromatography.

Quantitative High Performance Thin Layer Chromatography (HPTLC) requires the application of several standards to each plate, reducing the number of actual samples that can be analyzed in a single run. Using pure standard compounds and a selective detection method, the standards for quantitation can be applied besides - à côté - the chromatography area. This frees the sample application space to accommodate the maximal number of sample on each plate. Also, analysis time is spent exclusively on samples, drastically shortening the effective analysis time per sample and increasing sample throughput. Using this new calibration approach, the sample capacity of regular HPTLC methods can be increased or their scope be extended by an additional quantitative analysis. As a limitation, changes to the distribution of samples and standards within the plate as well as interferences from matrix compounds must be observed. We demonstrate the feasibility of this method by complementing an HPTLC method with a quantitative analysis of total anthocyanin content in colored wheat varieties. The quantitation was validated and compared to the conventional photometric analysis. As outcome, the additional photometric analysis could be replaced and rendered unnecessary, saving time, effort and equipment. This approach could also be employed to quantify highly retained substances, which are usually inaccessible for quantitative analysis.

Preparation and analytical characterisation of pure fractions of cellooligosaccharides.

In the present work, a viable protocol was developed to prepare monodisperse cellooligomers up to a degree of polymerisation (DP) of 20. Peracetylated cellooligosaccharides were obtained from cellulose by acetolysis and subsequently purified by Normal Phase-High Performance Liquid Chromatography using toluene, ethyl acetate and acetone as eluents. In addition, we demonstrated how to efficiently monitor the purity and dispersity of the obtained compounds by High Performance Thin Layer Chromatography, Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry and Nuclear Magnetic Resonance. With this approach, it is possible to isolate cellooligomer standards up to DP 20 on a preparative scale (dozens of mg). The column chromatographic separation proved to be robust over several months and to be scalable from a analytical to a preparative column. The isolated oligomer standards allow a more precise description of cellooligomer distributions typically emerging from biorefinery process streams after hydrolysis of lignocelluloses. They can be used to calibrate the oligomeric region in size-exclusion chromatography where light scattering detection fails due to limited scattering intensities.