Contrasting patterns of 5S rDNA repeats in European and Asian ecotypes of greater duckweed, Spirodela polyrhiza (Lemnaceae).

Ribosomal DNA (rDNA) contains highly conserved, specifically organized sequences encoding ribosomal RNAs (rRNAs) separated by variable non-transcribed intergenic spacers (NTSs) and is abundant in eukaryotic genomes. These characteristics make the rDNA an informative molecular target to study genome organization, molecular evolution, and phylogenetics. In this study, we characterized the 5S rDNA repeats in the greater duckweed Spiroldela polyrhiza, a species known for its small size, rapid growth, highly conserved genome organization, and low mutation rate. Sequence analysis of at least 12 individually cloned PCR fragments containing the 5S rDNA units for each of six ecotypes that originated from Europe (Ukraine) and Asia (China) revealed two distinct types of 5S rDNA repeats containing NTSs of different lengths and nucleotide compositions. The shorter 5S rDNA repeat units had a highly homogeneous 400-bp NTS, with few ecotype- or region-specific single-nucleotide polymorphisms (SNPs). The longer 5S rDNA units had NTSs of 1056-1084 bp with characteristic intra- and inter-genomic variants due to specific SNPs and insertions/deletions of 4-15-bp DNA elements. We also detected significant variability in the ratio of short/long 5S rDNA variants between ecotypes of S. polyrhiza. The contrasting dynamics of the two types of 5S rDNA units, combined with the unusually low repeat copy number (for plants) in S. polyrhiza (46-220 copies per genome), shows that this species could serve as an excellent model for examining the mechanisms of concerted evolution and functional significance of rDNA variability.

Duckweeds for Phytoremediation of Polluted Water.

Tiny aquatic plants from the Lemnaceae family, commonly known as duckweeds, are often regarded as detrimental to the environment because of their ability to quickly populate and cover the surfaces of bodies of water. Due to their rapid vegetative propagation, duckweeds have one of the fastest growth rates among flowering plants and can accumulate large amounts of biomass in relatively short time periods. Due to the high yield of valuable biomass and ease of harvest, duckweeds can be used as feedstock for biofuels, animal feed, and other applications. Thanks to their efficient absorption of nitrogen- and phosphate-containing pollutants, duckweeds play an important role in the restorative ecology of water reservoirs. Moreover, compared to other species, duckweed species and ecotypes demonstrate exceptionally high adaptivity to a variety of environmental factors; indeed, duckweeds remove and convert many contaminants, such as nitrogen, into plant biomass. The global distribution of duckweeds and their tolerance of ammonia, heavy metals, other pollutants, and stresses are the major factors highlighting their potential for use in purifying agricultural, municipal, and some industrial wastewater. In summary, duckweeds are a powerful tool for bioremediation that can reduce anthropogenic pollution in aquatic ecosystems and prevent water eutrophication in a simple, inexpensive ecologically friendly way. Here we review the potential for using duckweeds in phytoremediation of several major water pollutants: mineral nitrogen and phosphorus, various organic chemicals, and heavy metals.

Ammonium Uptake, Mediated by Ammonium Transporters, Mitigates Manganese Toxicity in Duckweed, Spirodela polyrhiza.

Nitrogen is an essential nutrient that affects all aspects of the growth, development and metabolic responses of plants. Here we investigated the influence of the two major sources of inorganic nitrogen, nitrate and ammonium, on the toxicity caused by excess of Mn in great duckweed, Spirodela polyrhiza. The revealed alleviating effect of ammonium on Mn-mediated toxicity, was complemented by detailed molecular, biochemical and evolutionary characterization of the species ammonium transporters (AMTs). Four genes encoding AMTs in S. polyrhiza, were classified as SpAMT1;1, SpAMT1;2, SpAMT1;3 and SpAMT2. Functional testing of the expressed proteins in yeast and Xenopus oocytes clearly demonstrated activity of SpAMT1;1 and SpAMT1;3 in transporting ammonium. Transcripts of all SpAMT genes were detected in duckweed fronds grown in cultivation medium, containing a physiological or 50-fold elevated concentration of Mn at the background of nitrogen or a mixture of nitrate and ammonium. Each gene demonstrated an individual expression pattern, revealed by RT-qPCR. Revealing the mitigating effect of ammonium uptake on manganese toxicity in aquatic duckweed S. polyrhiza, the study presents a comprehensive analysis of the transporters involved in the uptake of ammonium, shedding a new light on the interactions between the mechanisms of heavy metal toxicity and the regulation of the plant nitrogen metabolism.

Biodiversity of Duckweed (Lemnaceae) in Water Reservoirs of Ukraine and China Assessed by Chloroplast DNA Barcoding.

Monitoring and characterizing species biodiversity is essential for germplasm preservation, academic studies, and various practical applications. Duckweeds represent a group of tiny aquatic plants that include 36 species divided into 5 genera within the Lemnaceae family. They are an important part of aquatic ecosystems worldwide, often covering large portions of the water reservoirs they inhabit, and have many potential applications, including in bioremediation, biofuels, and biomanufacturing. Here, we evaluated the biodiversity of duckweeds in Ukraine and Eastern China by characterizing specimens using the two-barcode protocol with the chloroplast atpH-atpF and psbK-psbI spacer sequences. In total, 69 Chinese and Ukrainian duckweed specimens were sequenced. The sequences were compared against sequences in the NCBI database using BLAST. We identified six species from China (Spirodela polyrhiza, Landoltia punctata, Lemna aequinoctialis, Lemna minor, Lemna turionifera, and Wolffia globosa) and six from Ukraine (S. polyrhiza, Lemna gibba, Lemna minor, Lemna trisulca, Lemna turionifera, and Wolffia arrhiza). The most common duckweed species in the samples from Ukraine were Le. minor and S. polyrhiza, accounting for 17 and 15 out of 40 specimens, respectively. The most common duckweed species in the samples from China was S. polyrhiza, accounting for 15 out of 29 specimens. La. punctata and Le. aequinoctialis were also common in China, accounting for five and four specimens, respectively. According to both atpH-atpF and psbK-psbI barcode analyses, the species identified as Le. aequinoctialis does not form a uniform taxon similar to other duckweed species, and therefore the phylogenetic status of this species requires further clarification. By monitoring duckweeds using chloroplast DNA sequencing, we not only precisely identified local species and ecotypes, but also provided background for further exploration of native varieties with diverse genetic backgrounds. These data could be useful for future conservation, breeding, and biotechnological applications.

The Ribosomal DNA Loci of the Ancient Monocot Pistia stratiotes L. (Araceae) Contain Different Variants of the 35S and 5S Ribosomal RNA Gene Units.

The freshwater plant water lettuce (Pistia stratiotes L.) grows in warm climatic zones and is used for phytoremediation and biomass production. P. stratiotes belongs to the Araceae, an ecologically and structurally diverse early monocot family, but the phylogenetic relationships among Araceae members are poorly understood. Ribosomal DNAs (rDNAs), including the 35S and 5S rDNA, encode the RNA components of ribosomes and are widely used in phylogenetic and evolutionary studies of various plant taxa. Here, we comprehensively characterized the chromosomal locations and molecular organization of 35S and 5S rDNA genes in water lettuce using karyological and molecular methods. Fluorescence in situ hybridization revealed a single location for the 35S and 5S rDNA loci, each on a different pair of the species' 28 chromosomes. Molecular cloning and nucleotide sequencing of 35S rDNA of P. stratiotes, the first representative Araceae sensu stricto in which such a study was performed, displayed typical structural characteristics. The full-length repeat showed high sequence conservation of the regions producing the 18S, 5.8S, and 25S rRNAs and divergence of the internal transcribed spacers ITS1 and ITS2 as well as the large intergenic spacer (IGS). Alignments of the deduced sequence of 18S rDNA with the sequences available for other Araceae and representatives of other clades were used for phylogenetic analysis. Examination of 11 IGS sequences revealed significant intra-genomic length variability due to variation in subrepeat number, with four types of units detected within the 35S rDNA locus of the P. stratiotes genome (estimated size 407 Mb/1C). Similarly, the 5S rDNA locus harbors gene units comprising a conserved 119-bp sequence encoding 5S rRNA and two types of non-transcribed spacer (NTS) sequences. Type I was classified into four subtypes, which apparently originated via progressive loss of subrepeats within the duplicated NTS region containing the 3' part of the 5S rRNA gene. The minor Type II NTS is shorter than Type I and differs in nucleotide composition. Some DNA clones containing two or three consecutive 5S rDNA repeats harbored 5S rDNA genes with different types of NTSs, confirming the mosaic composition of the 5S rDNA locus.

Mosaic Arrangement of the 5S rDNA in the Aquatic Plant Landoltia punctata (Lemnaceae).

Duckweeds are a group of monocotyledonous aquatic plants in the Araceae superfamily, represented by 37 species divided into five genera. Duckweeds are the fastest growing flowering plants and are distributed around the globe; moreover, these plants have multiple applications, including biomass production, wastewater remediation, and making pharmaceutical proteins. Dotted duckweed (Landoltia punctata), the sole species in genus Landoltia, is one of the most resilient duckweed species. The ribosomal DNA (rDNA) encodes the RNA components of ribosomes and represents a significant part of plant genomes but has not been comprehensively studied in duckweeds. Here, we characterized the 5S rDNA genes in L. punctata by cloning and sequencing 25 PCR fragments containing the 5S rDNA repeats. No length variation was detected in the 5S rDNA gene sequence, whereas the nontranscribed spacer (NTS) varied from 151 to 524 bp. The NTS variants were grouped into two major classes, which differed both in nucleotide sequence and the type and arrangement of the spacer subrepeats. The dominant class I NTS, with a characteristic 12-bp TC-rich sequence present in 3-18 copies, was classified into four subclasses, whereas the minor class II NTS, with shorter, 9-bp nucleotide repeats, was represented by two identical sequences. In addition to these diverse subrepeats, class I and class II NTSs differed in their representation of cis-elements and the patterns of predicted G-quadruplex structures, which may influence the transcription of the 5S rDNA. Similar to related duckweed species in the genus Spirodela, L. punctata has a relatively low rDNA copy number, but in contrast to Spirodela and the majority of other plants, the arrangement of the 5S rDNA units demonstrated an unusual, heterogeneous pattern in L. punctata, as revealed by analyzing clones containing double 5S rDNA neighboring units. Our findings may further stimulate the research on the evolution of the plant rDNA and discussion of the molecular forces driving homogenization of rDNA repeats in concerted evolution.

Survey of Drought-Associated TAWRKY2-D1 Gene Diversity in Bread Wheat and Wheat Relatives.

Recent advances in plant genomics revealed numerous factors related to drought tolerance, including a family of WRKY transcription factors. The aim of this study was to evaluate polymorphism of the TaWRKY2-D1 across a range of bread wheat cultivars, interspecific hybrids, and wild wheat relatives within the Triticum genus as a potential molecular target for marker-assistant selection. The initial sequencing of the TaWRKY2-D1 gene in six Ukrainian commercial cultivars detected some sequence variations along the ~ 1.8 kb of gene promoter and the followed coding region composed of four exons and three introns. Based on the gained sequence information, five sets of primers covering different gene regions were designed to annotate theTaWRKY2-D1 genetic diversity in 202 wheat cultivars, including 77 accessions from the CIMMYT collection, 72 commercial varieties cultivated in Ukraine, and 53 hybrids and wild wheat species. The combination of developed DNA markers enabled effective and reproducible annotation of cultivars genetic diversity. The primers set targeting introns adjusted to the gene's exon 3, turned out to be the most informative for screening heterogeneity of the TaWRKY2-D1. The developed molecular markers represent effective, informative means for selecting drought tolerance germplasm donors to promote wheat breeding programs.

The Dynamics of NO3- and NH4+ Uptake in Duckweed Are Coordinated with the Expression of Major Nitrogen Assimilation Genes.

Duckweed plants play important roles in aquatic ecosystems worldwide. They rapidly accumulate biomass and have potential uses in bioremediation of water polluted by fertilizer runoff or other chemicals. Here we studied the assimilation of two major sources of inorganic nitrogen, nitrate (NO3- ) and ammonium (NH4+), in six duckweed species: Spirodela polyrhiza, Landoltia punctata, Lemna aequinoctialis, Lemna turionifera, Lemna minor, and Wolffia globosa. All six duckweed species preferred NH4+ over NO3- and started using NO3- only when NH4+ was depleted. Using the available genome sequence, we analyzed the molecular structure and expression of eight key nitrogen assimilation genes in S. polyrhiza. The expression of genes encoding nitrate reductase and nitrite reductase increased about 10-fold when NO3- was supplied and decreased when NH4+ was supplied. NO3- and NH4+ induced the glutamine synthetase (GS) genes GS1;2 and the GS2 by 2- to 5-fold, respectively, but repressed GS1;1 and GS1;3. NH4+ and NO3- upregulated the genes encoding ferredoxin- and NADH-dependent glutamate synthases (Fd-GOGAT and NADH-GOGAT). A survey of nitrogen assimilation gene promoters suggested complex regulation, with major roles for NRE-like and GAATC/GATTC cis-elements, TATA-based enhancers, GA/CTn repeats, and G-quadruplex structures. These results will inform efforts to improve bioremediation and nitrogen use efficiency.

Gene editing to facilitate hybrid crop production.

Capturing heterosis (hybrid vigor) is a promising way to increase productivity in many crops; hybrid crops often have superior yields, disease resistance, and stress tolerance compared with their parental inbred lines. The full utilization of heterosis faces a number of technical problems related to the specifics of crop reproductive biology, such as difficulties with generating and maintaining male-sterile lines and the low efficiency of natural cross-pollination for some genetic combinations. Innovative technologies, such as development of artificial in vitro systems for hybrid production and apomixis-based systems for maintenance of the resulting heterotic progeny, may substantially facilitate the production of hybrids. Genome editing using specifically targeted nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (CRISPR/Cas9) systems, which recognize targets by RNA:DNA complementarity, has recently become an integral part of research and development in life science. In this review, we summarize the progress of genome editing technologies for facilitating the generation of mutant male sterile lines, applications of haploids for hybrid production, and the use of apomixis for the clonal propagation of elite hybrid lines.

Chromosome-scale genome assembly for the duckweed Spirodela intermedia, integrating cytogenetic maps, PacBio and Oxford Nanopore libraries.

Duckweeds are small, free-floating, morphologically highly reduced organisms belonging to the monocot order Alismatales. They display the most rapid growth among flowering plants, vary ~ 14-fold in genome size and comprise five genera. Spirodela is the phylogenetically oldest genus with only two mainly asexually propagating species: S. polyrhiza (2n = 40; 160 Mbp/1C) and S. intermedia (2n = 36; 160 Mbp/1C). This study combined comparative cytogenetics and de novo genome assembly based on PacBio, Illumina and Oxford Nanopore (ON) reads to obtain the first genome reference for S. intermedia and to compare its genomic features with those of the sister species S. polyrhiza. Both species' genomes revealed little more than 20,000 putative protein-coding genes, very low rDNA copy numbers and a low amount of repetitive sequences, mainly Ty3/gypsy retroelements. The detection of a few new small chromosome rearrangements between both Spirodela species refined the karyotype and the chromosomal sequence assignment for S. intermedia.

Overexpression of the TaSHN1 transcription factor in bread wheat leads to leaf surface modifications, improved drought tolerance, and no yield penalty under controlled growth conditions.

Transcription factors regulate multiple networks, mediating the responses of organisms to stresses, including drought. Here, we investigated the role of the wheat transcription factor TaSHN1 in crop growth and drought tolerance. TaSHN1, isolated from bread wheat, was characterized for molecular interactions and functionality. The overexpression of TaSHN1 in wheat was followed by the evaluation of T2 and T3 transgenic lines for drought tolerance, growth, and yield components. Leaf surface changes were analysed by light microscopy, SEM, TEM, and GC-MS/GC-FID. TaSHN1 behaves as a transcriptional activator in a yeast transactivation assay and binds stress-related DNA cis-elements, determinants of which were revealed using 3D molecular modelling. The overexpression of TaSHN1 in transgenic wheat did not result in a yield penalty under the controlled plant growth conditions of a glasshouse. Transgenic lines had significantly lower stomatal density and leaf water loss and exhibited improved recovery after severe drought, compared with control plants. The comparative analysis of cuticular waxes revealed an increased accumulation of alkanes in leaves of transgenic lines. Our data demonstrate that TaSHN1 may operate as a positive modulator of drought stress tolerance. Positive attributes could be mediated through an enhanced accumulation of alkanes and reduced stomatal density.