Improved Protocols of ITS1-Based Metabarcoding and Their Application in the Analysis of Plant-Containing Products.

Plants are widely used for food and beverage preparation, most often in the form of complex mixtures of dried and ground parts, such as teas, spices or herbal medicines. Quality control of such products is important due to the potential health risks from the presence of unlabelled components or absence of claimed ones. A promising approach to analyse such products is DNA metabarcoding due to its high resolution and sensitivity. However, this method's application in food analysis requires several methodology optimizations in DNA extraction, amplification and library preparation. In this study, we present such optimizations. The most important methodological outcomes are the following: 1) the DNA extraction method greatly influences amplification success; 2) the main problem for the application of metabarcoding is DNA purity, not integrity or quantity; and 3) the "non-amplifiable" samples can be amplified with polymerases resistant to inhibitors. Using this optimized workflow, we analysed a broad set of plant products (teas, spices and herbal remedies) using two NGS platforms. The analysis revealed the problem of both the presence of extraneous components and the absence of labelled ones. Notably, for teas, no correlation was found between the price and either the absence of labelled components or presence of unlabelled ones; for spices, a negative correlation was found between the price and presence of unlabelled components.

Arabidopsis DNA topoisomerase I alpha is required for adaptive response to light and flower development.

DNA topoisomerase I alpha (TOP1α) plays a specific role in Arabidopsis thaliana development and is required for stem cell regulation in shoot and floral meristems. Recently, a new role independent of meristem functioning has been described for TOP1α, namely flowering time regulation. The same feature had been detected by us earlier for fas5, a mutant allele of TOP1α In this study we clarify the effects of fas5 on bolting initiation and analyze the molecular basis of its role on flowering time regulation. We show that fas5 mutation leads to a constitutive shade avoidance syndrome, accompanied by leaf hyponasty, petiole elongation, lighter leaf color and early bolting. Other alleles of TOP1α demonstrate the same shade avoidance response. RNA sequencing confirmed the activation of shade avoidance gene pathways in fas5 mutant plants. It also revealed the repression of many genes controlling floral meristem identity and organ morphogenesis. Our research further expands the knowledge of TOP1α function in plant development and reveals that besides stem cell maintenance TOP1α plays an important new role in regulating the adaptive plant response to light stimulus and flower development.

Dimorphic DNA variation in the anionic peroxidase gene AtPrx53 of Arabidopsis thaliana.

The DNA polymorphism in the AtPrx53 gene which encodes anionic peroxidase was analyzed in 20 Arabidopsis thaliana accessions. There are two divergent sequence types (Col and Dj-like haplotypes) in the AtPrx53 gene that differ by 2 indel and 16 non-singleton nucleotide polymorphisms including 5 nucleotide polymorphic sites responsible for 4 deduced amino acid replacements. Two of the amino acid substitutions (Phe/Ser180and Asp/Asn270) could be responsible for the difference in electrophoretic mobility of AtPrx53 allozymes. One of them (Phe/Ser180) lies within the hypervariable region, indicating that this amino acid polymorphism is subjected to balancing selection. The revealed difference between deduced allozymes is related to the dimorphism in mobility of three major anionic peroxidase isoforms which according to previously established data encoded by AtPrx53 gene. The haplotype Col which included 12 accessions from three different continents is characterized by faster mobility of three isoforms in comparison with the Dj haplotype represented by eight accessions. There is a significant association between the haplotype and several developmental traits: leaf number, flowering time, main stem height etc. Lines of the Dj haplotype have shorter duration of vegetative stages and flower earlier than most of Col haplotype accessions. The reasons of this association are discussed.