A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L.

Root chicory (Cichorium intybus L. var. sativum) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associated costs, recently chicory variants with a lower sesquiterpene lactone content were created by inactivating the four copies of the germacrene A synthase gene (CiGAS-S1, -S2, -S3, -L) which encode the enzyme initiating bitter sesquiterpene lactone biosynthesis in chicory. In this study, different delivery methods for CRISPR/Cas9 reagents have been compared regarding their efficiency to induce mutations in the CiGAS genes, the frequency of off-target mutations as well as their environmental and economic impacts. CRISPR/Cas9 reagents were delivered by Agrobacterium-mediated stable transformation or transient delivery by plasmid or preassembled ribonucleic complexes (RNPs) using the same sgRNA. All methods used lead to a high number of INDEL mutations within the CiGAS-S1 and CiGAS-S2 genes, which match the used sgRNA perfectly; additionally, the CiGAS-S3 and CiGAS-L genes, which have a single mismatch with the sgRNA, were mutated but with a lower mutation efficiency. While using both RNPs and plasmids delivery resulted in biallelic, heterozygous or homozygous mutations, plasmid delivery resulted in 30% of unwanted integration of plasmid fragments in the genome. Plants transformed via Agrobacteria often showed chimerism and a mixture of CiGAS genotypes. This genetic mosaic becomes more diverse when plants were grown over a prolonged period. While the genotype of the on-targets varied between the transient and stable delivery methods, no off-target activity in six identified potential off-targets with two to four mismatches was found. The environmental impacts (greenhouse gas (GHG) emissions and primary energy demand) of the methods are highly dependent on their individual electricity demand. From an economic view - like for most research and development activities - employment and value-added multiplier effects are high; particularly when compared to industrial or manufacturing processes. Considering all aspects, we conclude that using RNPs is the most suitable method for genome editing in chicory since it led to a high efficiency of editing, no off-target mutations, non-transgenic plants with no risk of unwanted integration of plasmid DNA and without needed segregation of transgenes.

New Insights Into the Activity of Apple Dihydrochalcone Phloretin: Disturbance of Auxin Homeostasis as Physiological Basis of Phloretin Phytotoxic Action.

Apple species are the unique naturally rich source of dihydrochalcones, phenolic compounds with an elusive role in planta, but suggested auto-allelochemical features related to "apple replant disease" (ARD). Our aim was to elucidate the physiological basis of the phytotoxic action of dihydrochalcone phloretin in the model plant Arabidopsis and to promote phloretin as a new prospective eco-friendly phytotoxic compound. Phloretin treatment induced a significant dose-dependent growth retardation and severe morphological abnormalities and agravitropic behavior in Arabidopsis seedlings. Histological examination revealed a reduced starch content in the columella cells and a serious disturbance in root architecture, which resulted in the reduction in length of meristematic and elongation zones. Significantly disturbed auxin metabolome profile in roots with a particularly increased content of IAA accumulated in the lateral parts of the root apex, accompanied by changes in the expression of auxin biosynthetic and transport genes, especially PIN1, PIN3, PIN7, and ABCB1, indicates the role of auxin in physiological basis of phloretin-induced growth retardation. The results reveal a disturbance of auxin homeostasis as the main mechanism of phytotoxic action of phloretin. This mechanism makes phloretin a prospective candidate for an eco-friendly bioherbicide and paves the way for further research of phloretin role in ARD.

The Arabinogalactan Protein Family of Centaurium erythraea Rafn.

Centaurium erythraea (centaury) is a medicinal plant with exceptional developmental plasticity in vitro and vigorous, often spontaneous, regeneration via shoot organogenesis and somatic embryogenesis, during which arabinogalactan proteins (AGPs) play an important role. AGPs are highly glycosylated proteins belonging to the super family of O-glycosylated plant cell surface hydroxyproline-rich glycoproteins (HRGPs). HRGPs/AGPs are intrinsically disordered and not well conserved, making their homology-based mining ineffective. We have applied a recently developed pipeline for HRGP/AGP mining, ragp, which is based on machine learning prediction of proline hydroxylation, to identify HRGP sequences in centaury transcriptome and to classify them into motif and amino acid bias (MAAB) classes. AGP sequences with low AG glycomotif representation were also identified. Six members of each of the three AGP subclasses, fasciclin-like AGPs, receptor kinase-like AGPs and AG peptides, were selected for phylogenetic and expression analyses. The expression of these 18 genes was recorded over 48 h following leaf mechanical wounding, as well as in 16 tissue samples representing plants from nature, plants cultivated in vitro, and developmental stages during shoot organogenesis and somatic embryogenesis. None of the selected genes were upregulated during both wounding recovery and regeneration. Possible functions of AGPs with the most interesting expression profiles are discussed.

Secondary Somatic Embryogenesis in Centaurium erythraea Rafn.

Somatic embryogenesis (SE) is a developmental process during which plant somatic cells, under suitable conditions, produce embryogenic cells that develop into somatic embryos (se). SE is the most important method for plant propagation in vitro, having both fundamental and applicative significance. SE can be induced from different tissues and organs, but when se are used as explants, the process is recognized as secondary or cyclic SE. We induced secondary SE in Centaurium erythraea by application of 2,4-dichlorophenoxyacetic acid (2,4-D) and N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU). A medium containing 0.1 mgL-1 2,4-D and 0.25 mgL-1 CPPU was optimal in terms of the number of primary SE explants forming se, the number of well-developed se per explant, and morphological appearance of the obtained se. These concentrations allowed SE to progress through three cycles, whereas at higher concentrations of 0.2 mgL-1 2,4-D and 0.5 mgL-1 CPPU, only two cycles were achieved. Histological analysis revealed that secondary se are formed both directly and indirectly. Secondary SE readily germinated and converted into plantlets. Induction of cyclic SE contributes to the conservation efforts of this endangered medicinal plant and expands the spectrum of in vitro developmental pathways described in centaury-an emerging model in developmental biology.

Somatic Embryogenesis in Centaurium erythraea Rafn-Current Status and Perspectives: A Review.

Centaurium erythraea (centaury) is a traditionally used medicinal plant, with a spectrum of secondary metabolites with confirmed healing properties. Centaury is an emerging model in plant developmental biology due to its vigorous regenerative potential and great developmental plasticity when cultured in vitro. Hereby, we review nearly two decades of research on somatic embryogenesis (SE) in centaury. During SE, somatic cells are induced by suitable culture conditions to express their totipotency, acquire embryogenic characteristics, and eventually give rise to somatic embryos. When SE is initiated from centaury root explants, the process occurs spontaneously (on hormone-free medium), directly (without the callusing phase), and the somatic embryos are of unicellular origin. SE from leaf explants has to be induced by plant growth regulators and is indirect (preceded by callusing). Histological observations and culture conditions are compared in these two systems. The changes in antioxidative enzymes were followed during SE from the leaf explants. Special focus is given to the role of arabinogalactan proteins during SE, which were analyzed using a variety of approaches. The newest and preliminary results, including centaury transcriptome, novel potential SE markers, and novel types of arabinogalactan proteins, are discussed as perspectives of centaury research.

Silencing of germacrene A synthase genes reduces guaianolide oxalate content in Cichorium intybus L.

Chicory (Cichorium intybus L.) is a medicinal and industrial plant from the Asteraceae family that produces a variety of sesquiterpene lactones (STLs), most importantly bitter guaianolides: lactucin, lactucopicrin and 8-deoxylactucin as well as their modified forms such as oxalates. These compounds have medicinal properties; however, they also hamper the extraction of inulin - a very important food industry product from chicory roots. The first step in guaianolide biosynthesis is catalyzed by germacrene A synthase (GAS) which in chicory exists in two isoforms - GAS long (encoded by CiGASlo) and GAS short (encoded by CiGASsh). AmiRNA silencing was used to obtain plants with reduced GAS gene expression and level of downstream metabolites, guaianolide-15-oxalates, as the major STLs in chicory. This approach could be beneficial for engineering new chicory varieties with varying STL content, and especially varieties with reduced bitter compounds more suitable for inulin production.

ragp: Pipeline for mining of plant hydroxyproline-rich glycoproteins with implementation in R.

Hydroxyproline-rich glycoproteins (HRGPs) are one of the most complex families of macromolecules found in plants, due to the diversity of glycans decorating the protein backbone, as well as the heterogeneity of the protein backbones. While this diversity is responsible for a wide array of physiological functions associated with HRGPs, it hinders attempts for homology based identification. Current approaches, based on identifying sequences with characteristic motifs and biased amino acid composition, are limited to prototypical sequences. Ragp is an R package for mining and analysis of HRGPs, with emphasis on arabinogalactan proteins. The ragp filtering pipeline exploits one of the HRGPs key features, the presence of hydroxyprolines which represent glycosylation sites. Main package features include prediction of proline hydroxylation sites, amino acid motif and bias analyses, efficient communication with web servers for prediction of N-terminal signal peptides, glycosylphosphatidylinositol modification sites and disordered regions and the ability to annotate sequences through hmmscan and subsequent GO enrichment, based on predicted Pfam domains. As such, ragp extends R's rich ecosystem for high-throughput sequence data analyses. The ragp R package is available under the MIT Open Source license and is freely available to download from GitHub at: https://github.com/missuse/ragp.

Knockout mutants as a tool to identify the subunit composition of Arabidopsis glutamine synthetase isoforms.

Glutamine synthetase (GS) is a key enzyme in nitrogen assimilation, which catalyzes the formation of glutamine from ammonia and glutamate. Plant GS isoforms are multimeric enzymes, recently shown to be decamers. The Arabidopsis genome encodes five cytosolic (GS1) proteins labeled as GLN1;1 through GLN1;5 and one chloroplastic (GS2) isoform, GLN2;0. However, as many as 11 GS activity bands were resolved from different Arabidopsis tissues by Native PAGE and activity staining. Western analysis showed that all 11 isoforms are composed exclusively of 40 kDa GS1 subunits. Of five GS1 genes, only GLN1;1, GLN1;2 and GLN1;3 transcripts accumulated to significant levels in vegetative tissues, indicating that only subunits encoded by these three genes produce the 11-band zymogram. Even though the GS2 gene also had significant expression, the corresponding activity was not detected, probably due to inactivation. To resolve the subunit composition of 11 active GS1 isoforms, homozygous knockout mutants deficient in the expression of different GS1 genes were selected from the progeny of T-DNA insertional SALK and SAIL lines. Comparison of GS isoenzyme patterns of the selected GS1 knockout mutants indicated that all of the detected isoforms consist of varying proportions of GLN1;1, GLN1;2 and GLN1;3 subunits, and that GLN1;1 and GLN1;3, as well as GLN1;2 and GLN1;3 and possibly GLN1;1 and GLN1;2 proteins combine in all proportions to form active homo- and heterodecamers.

Herbicide phosphinothricin causes direct stimulation hormesis.

Herbicide phosphinothricin (PPT) inhibits glutamine synthetase (GS), a key enzyme in nitrogen assimilation, thus causing ammonia accumulation, glutamine depletion and eventually plant death. However, the growth response of Lotus corniculatus L. plants immersed in solutions with a broad range of PPT concentrations is biphasic, with pronounced stimulating effect on biomass production at concentrations ≤ 50 µM and growth inhibition at higher concentrations. The growth stimulation at low PPT concentrations is a result of activation of chloroplastic isoform GS2, while the growth suppression is caused by inhibition of both cytosolic GS1 and GS2 at higher PPT concentrations. Since the results are obtained in cell-free system (e.g. protein extracts), to which the principles of homeostasis are not applicable, this PPT effect is an unambiguous example of direct stimulation hormesis. A detailed molecular mechanism of concentration-dependent interaction of both PPT and a related GS inhibitor, methionine sulfoximine, with GS holoenzymes is proposed. The mechanism is in concurrence with all experimental and literature data.