Induced Chirality in Canthaxanthin Aggregates Reveals Multiple Levels of Supramolecular Organization.

Carotenoids tend to form supramolecular aggregates via non-covalent interactions where the chirality of individual molecules is amplified to the macroscopic level. We show that this can also be achieved for non-chiral carotenoid monomers interacting with polysaccharides. The chirality induction in canthaxanthin (CAX), caused by heparin (HP) and hyaluronic acid (HA), was monitored by chiroptical spectroscopy. Electronic circular dichroism (ECD) and Raman optical activity (ROA) spectra indicated the presence of multiple carotenoid formations, such as H- and J-type aggregates. This is consistent with molecular dynamics (MD) and density functional theory (DFT) simulations of the supramolecular structures and their spectroscopic response.

Comparative analysis of the carrot miRNAome in response to salt stress.

Soil salinity adversely affects the yield and quality of crops, including carrot. During salt stress, plant growth and development are impaired by restricted water uptake and ion cytotoxicity, leading to nutrient imbalance and oxidative burst. However, the molecular mechanisms of the carrot plant response to salt stress remain unclear. The occurrence and expression of miRNAs that are potentially involved in the regulation of carrot tolerance to salinity stress were investigated. The results of small RNA sequencing revealed that salt-sensitive (DH1) and salt-tolerant (DLBA) carrot varieties had different miRNA expression profiles. A total of 95 miRNAs were identified, including 71 novel miRNAs, of which 30 and 23 were unique to DH1 and DLBA, respectively. The comparison of NGS and qPCR results allowed identification of two conserved and five novel miRNA involved in carrot response to salt stress, and which differentiated the salt-tolerant and salt-sensitive varieties. Degradome analysis supported by in silico-based predictions and followed by expression analysis of exemplary target genes pointed at genes related to proline, glutathione, and glutamate metabolism pathways as potential miRNA targets involved in salt tolerance, and indicated that the regulation of osmoprotection and antioxidant protection, earlier identified as being more efficient in the tolerant variety, may be controlled by miRNAs. Furthermore, potential miRNA target genes involved in chloroplast protection, signal transduction and the synthesis and modification of cell wall components were indicated in plants growing in saline soil.

The Impact of Gold Nanoparticles on Somatic Embryogenesis Using the Example of Arabidopsis thaliana.

Although the influence of nanoparticles (NPs) on developmental processes is better understood, little is known about their impact on somatic embryogenesis (SE). This process involves changes in the direction of cell differentiation. Thus, studying the effect of NPs on SE is essential to reveal their impact on cell fate. This study aimed to examine the influence of gold nanoparticles (Au NPs) with different surface charges on the SE of 35S:BBM Arabidopsis thaliana, with particular emphasis on the spatiotemporal localization of pectic arabinogalactan proteins (AGPs) and extensin epitopes in cells changing the direction of their differentiation. The results show that under the influence of nanoparticles, the explant cells of 35S:BBM Arabidopsis thaliana seedling origin did not enter the path of SE. Bulges and the formation of organ-like structures were observed in these explants, in contrast to the control, where somatic embryos developed. Additionally, spatiotemporal changes in the chemical composition of the cell walls during the culture were observed. Under the influence of Au NPs, the following effects were observed: (1) explant cells did not enter the SE pathway, (2) the impacts of Au NPs with different surface charges on the explants were variable, and (3) the compositions of the analyzed pectic AGPs and extensin epitopes were diverse in the cells with different developmental programs: SE (control) and non-SE (treated with Au NPs).

Antioxidative and osmoprotecting mechanisms in carrot plants tolerant to soil salinity.

Soil salinization is a growing problem for agriculture worldwide and carrot is one the most salt-sensitive vegetable species. However, some varieties are capable of withstanding high salt concentrations due to unknown genetic and physiological mechanisms. The aim of this work was to reveal protecting mechanisms against osmotic and ionic stresses that contribute to salt tolerance in carrot. For this purpose, changes in biochemical traits due to soil salinity occurring in the salt-tolerant and salt-sensitive plants were determined. The obtained results showed that the tolerance of the salt-tolerant variety was partially determined constitutively, however, the exposition to saline soil triggered a physiological response that was more evident in the root than in the leaves. The most noticeable changes were the high increase in the content of osmoprotective proline and other low molecular antioxidants such as glutathione and ascorbic acid, and the decrease in the ratio of reduced to oxidized glutathione forms. These changes imply an efficient operation of the ascorbate-glutathione cycle that together with a high activity of antioxidative enzymes such as peroxidases, indicate on the induction of mechanisms associated mainly with protection against excessive reactive oxygen species.

Multiplex Site-Directed Gene Editing Using Polyethylene Glycol-Mediated Delivery of CRISPR gRNA:Cas9 Ribonucleoprotein (RNP) Complexes to Carrot Protoplasts.

The aim of this work was to show an efficient, recombinant DNA-free, multiplex gene-editing method using gRNA:Cas9 ribonucleoprotein (RNP) complexes delivered directly to plant protoplasts. For this purpose, three RNPs were formed in the tube, their activity was confirmed by DNA cleavage in vitro, and then they were delivered to carrot protoplasts incubated with polyethylene glycol (PEG). After 48 h of incubation, single nucleotide deletions and insertions and small deletions at target DNA sites were identified by using fluorescent-PCR capillary electrophoresis and sequencing. When two or three RNPs were delivered simultaneously, long deletions of 33-152 nt between the gRNA target sites were generated. Such mutations occurred with an efficiency of up to 12%, while the overall editing effectiveness was very high, reaching 71%. This highly efficient multiplex gene-editing method, without the need for recombinant DNA technology, can be adapted to other plants for which protoplast culture methods have been established.

Repression of Carotenoid Accumulation by Nitrogen and NH4+ Supply in Carrot Callus Cells In Vitro.

The effect of mineral nutrition on the accumulation of the main health beneficial compounds in carrots, the carotenoid pigments, remains ambiguous; here, a model-based approach was applied to reveal which compounds are responsible for the variation in carotenoid content in carrot cells in vitro. For this purpose, carotenoid-rich callus was cultured on either BI (modified Gamborg B5) or R (modified Murashige and Skoog MS) mineral media or on modified media obtained by exchanging compounds between BI and R. Callus growing on the BI medium had abundant carotene crystals in the cells and a dark orange color in contrast to pale orange callus with sparse crystals on the R medium. The carotenoid content, determined by HPLC and spectrophotometrically after two months of culture, was 5.3 higher on the BI medium. The replacement of media components revealed that only the N concentration and the NO3:NH4 ratio affected carotenoid accumulation. Either the increase of N amount above 27 mM or decrease of NO3:NH4 ratio below 12 resulted in the repression of carotenoid accumulation. An adverse effect of the increased NH4+ level on callus growth was additionally found. Somatic embryos were formed regardless of the level of N supplied. Changes to other media components, i.e., macroelements other than N, microelements, vitamins, growth regulators, and sucrose had no effect on callus growth and carotenoid accumulation. The results obtained from this model system expand the range of factors, such as N availability, composition of N salts, and ratio of nitrate to ammonium N form, that may affect the regulation of carotenoid metabolism.

Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot.

Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of psy paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two psy paralogs in a model system and to reveal biochemical changes in the cell wall of psy knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (Daucus carota) callus cells in order to induce mutations in the psy1 and psy2 genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the psy2 gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The psy2 knockout also decreased the expression of the psy1 paralog. Immunohistochemical staining of the psy2 mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free psy2 mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling.

Persistence of Xanthomonas campestris pv. campestris in Field Soil in Central Europe.

Xanthomonas campestris pv. campestris (Xcc) is a bacterium that causes black rot of crucifers. The greatest losses of brassica crop production usually result from seed-borne infection, but carry-over of inoculum in field soil may also be possible. The aim of this study was to monitor persistence of Xcc in field soil in central Europe using a conventional PCR assay with hrpF primers and a two-step nested real-time PCR assay using Zur primers. The work has demonstrated that nested real-time PCR can be used to improve the analytical sensitivity for detection of Xcc in soil compared to conventional PCR, and that Xcc may persist in soil for up to two years following an infected brassica crop in central European climatic conditions.

Electronic Circular Dichroism of the Cas9 Protein and gRNA:Cas9 Ribonucleoprotein Complex.

The Streptococcus pyogenes Cas9 protein (SpCas9), a component of CRISPR-based immune system in microbes, has become commonly utilized for genome editing. This nuclease forms a ribonucleoprotein (RNP) complex with guide RNA (gRNA) which induces Cas9 structural changes and triggers its cleavage activity. Here, electronic circular dichroism (ECD) spectroscopy was used to confirm the RNP formation and to determine its individual components. The ECD spectra had characteristic features differentiating Cas9 and gRNA, the former showed a negative/positive profile with maxima located at 221, 209 and 196 nm, while the latter revealed positive/negative/positive/negative pattern with bands observed at 266, 242, 222 and 209 nm, respectively. For the first time, the experimental ECD spectrum of the gRNA:Cas9 RNP complex is presented. It exhibits a bisignate positive/negative ECD couplet with maxima at 273 and 235 nm, and it differs significantly from individual spectrum of each RNP components. Additionally, the Cas9 protein and RNP complex retained biological activity after ECD measurements and they were able to bind and cleave DNA in vitro. Hence, we conclude that ECD spectroscopy can be considered as a quick and non-destructive method of monitoring conformational changes of the Cas9 protein as a result of Cas9 and gRNA interaction, and identification of the gRNA:Cas9 RNP complex.

Real-time detection of somatic hybrid cells during electrofusion of carrot protoplasts with stably labelled mitochondria.

Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.

Light Microscopy and Raman Imaging of Carotenoids in Plant Cells In Situ and in Released Carotene Crystals.

Light microscopy with a bright field mode offers an easy and fast examination of plant specimen for carotenoid presence in its cells. Using basic techniques such as hand sectioned or squashed preparations, carotenoid-rich chromoplasts can be identified without applying any staining procedure and their localization within the cell, their shape and number can be assessed. More detailed information can be obtained by using Raman spectroscopy which is suitable for the analysis of carotenoids due to their unique Raman spectra and allows semiquantification of their contents. Raman imaging (mapping) can be additionally used to show the distribution of carotenoids within the sample. Raman spectra can be taken from extracted carotenoids but can be also obtained directly from plant tissues or cells as Raman measurements are nondestructive for the sample. Here we describe preparations of intact tissue samples, monolayer cell samples, isolated protoplasts as well as carotene crystals released from chromoplasts that are suitable for subsequent observations using light microscopy and for analysis using Raman spectroscopy.

Agrobacterium rhizogenes-mediated transformation of Hypericum sinaicum L. for the development of hairy roots containing hypericin

Hypericum sinaicum L. is an endangered Egyptian medicinal plant of high importance due to the presence of naphthodianthrones (hypericins), which have photodynamic properties and pharmaceutical potential. We sought to assess H. sinaicum ability to develop hairy roots that could be cultured in contained conditions in vitro and used as a source for hypericin production. We used four A. rhizogenes strains differing in their plasmids and chromosomal backgrounds to inoculate excised H. sinaicum root, stem and leaf explants to induce hairy root development. Additionally, inoculum was applied to shoots held in Rockwool cubes supporting their stand after removal of the root system. All explant types were susceptible to A. rhizogenes although stem explants responded more frequently (over 90%) than other explant types. The A4 and A4T A. rhizogenes strains were highly, and equally effective in hairy root induction on 66-72% of explants while the LBA1334 strain was the most effective in transformation of shoots. Sonication applied to explants during inoculation enhanced the frequency of hairy root development, the most effective was 60 s treatment doubling the percentage of explants with hairy roots. However, shoot transformation was the most effective approach as shoots developed hairy roots within 10 days after inoculation. Molecular analyses confirmed that the established hairy root cultures in vitro were indeed obtained due to a horizontal gene transfer from bacteria. These cultures grew fast and the hypericin content in hairy roots was about two fold higher than in H. sinaicum plants as determined by HPLC.

Combined biofortification of carrot with iodine and selenium.

The aim of a three-year study was to assess the effect of combined biofortification with I and Se in carrot. Four cultivars ('Askona' F1, 'Samba' F1, 'Kazan' F1 and 'White Satin') were grown in soil fertilized with KI (4 kg I ha-1) and Na2SeO4 (0.25 kg Se ha-1). The I + Se fertilization did not affect yield but the plants of all cultivars accumulated both elements in leaves and roots. On average, the I and Se contents in roots increased 7.7-times for I and 4.9-times for Se as well as the average I:Se molar ratio was 0.28:1. The contents of both elements in roots remained well below the hazard threshold thus the intake of 100 g of biofortified carrot would substantially cover the RDA for I and Se. The changes in chemical composition of roots (nitrates, phenolic compounds, sugars, carotenoids, macro-, microelements and cadmium) were rather year-dependent than affected by the applied I + Se fertilization.

Chiral Amplification in Nature: Studying Cell-Extracted Chiral Carotenoid Microcrystals via the Resonance Raman Optical Activity of Model Systems.

Carotenoid microcrystals, extracted from cells of carrot roots and consisting of 95 % of achiral ß-carotene, exhibit a very intense chiroptical (ECD and ROA) signal. The preferential chirality of crystalline aggregates that consist mostly of achiral building blocks is a newly observed phenomenon in nature, and may be related to asymmetric information transfer from the chiral seeds (small amount of α-carotene or lutein) present in carrot cells. To confirm this hypothesis, we synthesized several model aggregates from various achiral and chiral carotenoids. Because of the sergeant-and-soldier behavior, a small number of chiral sergeants (α-carotene or astaxanthin) force the achiral soldier molecules (ß- or 11,11'-[D2 ]-ß-carotene) to jointly form supramolecular assemblies of induced chirality. The chiral amplification observed in these model systems confirmed that chiral microcrystals appearing in nature might consist predominantly of achiral building blocks and their supramolecular chirality might result from the co-crystallization of chiral and achiral analogues.

The development of a hairless phenotype in barley roots treated with gold nanoparticles is accompanied by changes in the symplasmic communication.

Uptake of water and nutrients by roots affects the ontogenesis of the whole plant. Nanoparticles, e.g. gold nanoparticles, have a broad range of applications in many fields which leads to the transfer of these materials into the environment. Thus, the understanding of their impact on the growth and development of the root system is an emerging issue. During our studies on the effect of positively charged gold nanoparticles on the barley roots, a hairless phenotype was found. We investigated whether this phenotype correlates with changes in symplasmic communication, which is an important factor that regulates, among others, differentiation of the rhizodermis into hair and non-hair cells. The results showed no restriction in symplasmic communication in the treated roots, in contrast to the control roots, in which the trichoblasts and atrichoblasts were symplasmically isolated during their differentiation. Moreover, differences concerning the root morphology, histology, ultrastructure and the cell wall composition were detected between the control and the treated roots. These findings suggest that the harmful effect of nanoparticles on plant growth may, among others, consist in disrupting the symplasmic communication/isolation, which leads to the development of a hairless root phenotype, thus limiting the functioning of the roots.

Visual Assay for Gene Editing Using a CRISPR/Cas9 System in Carrot Cells.

The development of the Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) system has advanced genome editing and has become widely adopted for this purpose in many species. Its efficient use requires the method adjustment and optimization. Here, we show the use of a model carrot callus system for demonstrating gene editing via CRISPR/Cas9 targeted mutagenesis. The system relies on the utilization of carrot tissue accumulating anthocyanin pigments responsible for a deep purple cell color and generation of knockout mutations in the flavanone-3-hydroxylase (F3H) gene in the anthocyanin biosynthesis pathway. F3H mutant cells targeted by Cas9/gRNA complexes are not able to synthesize anthocyanins and remain white, easily visually distinguished from purple wild-type cells. Mutations are either small indels or larger chromosomal deletions that can be identified by restriction fragment analysis and sequencing. This feasible system can also be applied for validating efficiency of CRISPR/Cas9 vectors.

Unique chromoplast organisation and carotenoid gene expression in carotenoid-rich carrot callus.

MAIN CONCLUSION: The new model orange callus line, similar to carrot root, was rich in carotenoids due to altered expression of some carotenogenesis-associated genes and possessed unique diversity of chromoplast ultrastructure. Callus induced from carrot root segments cultured in vitro is usually pale yellow (p-y) and poor in carotenoids. A unique, non-engineered callus line of dark orange (d-o) colour was developed in this work. The content of carotenoid pigments in d-o callus was at the same level as in an orange carrot storage root and nine-fold higher than in p-y callus. Carotenoids accumulated mainly in abundant crystalline chromoplasts that are also common in carrot root but not in p-y callus. Using transmission electron microscopy, other types of chromoplasts were also found in d-o callus, including membranous chromoplasts rarely identified in plants and not observed in carrot root until now. At the transcriptional level, most carotenogenesis-associated genes were upregulated in d-o callus in comparison to p-y callus, but their expression was downregulated or unchanged when compared to root tissue. Two pathway steps were critical and could explain the massive carotenoid accumulation in this tissue. The geranylgeranyl diphosphate synthase gene involved in the biosynthesis of carotenoid precursors was highly expressed, while the ß-carotene hydroxylase gene involved in ß-carotene conversion to downstream xanthophylls was highly repressed. Additionally, paralogues of these genes and phytoene synthase were differentially expressed, indicating their tissue-specific roles in carotenoid biosynthesis and metabolism. The established system may serve as a novel model for elucidating plastid biogenesis that coincides with carotenogenesis.

Haploid and Doubled Haploid Plant Production in Carrot Using Induced Parthenogenesis and Ovule Excision In Vitro.

Haploid plants have a gametophytic number of chromosomes (n) in the sporophyte. A doubled haploid (DH) plant results from doubling the chromosome set of a haploid plant, as a consequence a homozygosity plant is produced at every locus (true homozygous plant). DH plants are of great significance in breeding programs for the improvement of plants. Here we describe a protocol for the production of doubled haploid plants in carrot (Daucus carota L.) using parthenogenesis induced by wide pollination.

Organic iodine supply affects tomato plants differently than inorganic iodine.

Iodine is a beneficial element for humans but very lowly represented in our diet. Iodine-enriched vegetables could boost the iodine content in the food chain. Despite being a beneficial element for plants, little is known about the effect of different iodine forms on plant growth. This work analyses the effect of uptake of mineral (KI) and organoiodine (5-iodosalicylic acid, 5-ISA; 3,5-diiodosalicylic acid, 3,5-di-ISA; 2-iodobenzoic acid, 2-IBeA; 4-iodobenzoic acid, 4-IBeA) compounds on tomato plants at an early stage of vegetative growth. As many organoiodine compounds are derived from salicylic (SA) and benzoic acids (BeA), treatments with I, SA and BeA in various treatments were realized and the influence of tested compounds on plant growth was analyzed. Iodine content was measured, as well as expression of key genes involved in I and SA metabolism. Organoiodine compounds accumulated mainly in roots whereas iodine accumulated in the upper parts when given as KI. The shoot system had 5, 12 and 25 times higher iodine content after KI treatment than after 4-IBeA, 5-ISA and 2-IBeA, or 3,5-diISA treatments, respectively. A toxic effect on plants was observed only for 3,5-diISA and 4-IBeA. The expression levels of a gene related to iodine metabolism (HMT, halide ion methylotransferase), a gene responsible for SA methylation in leaves (SAMT) and a gene related to SA catabolism (S3H, salicylic acid 3-hydroxylase) were modified differently depending on the iodine source. Overall, our data point out to a difference in plant uptake, transport of iodine in tomato plants based on the form of iodine compound.

Raman, AFM and SNOM high resolution imaging of carotene crystals in a model carrot cell system.

Three non-destructive and complementary techniques, Raman imaging, Atomic Force Microscopy and Scanning Near-field Optical Microscopy were used simultaneously to show for the first time chemical and structural differences of carotenoid crystals. Spectroscopic and microscopic scanning probe measurements were applied to the released crystals or to crystals accumulated in a unique, carotenoids rich callus tissue growing in vitro that is considered as a new model system for plant carotenoid research. Three distinct morphological crystal types of various carotenoid composition were identified, a needle-like, rhomboidal and helical. Raman imaging using 532 and 488 nm excitation lines provided evidence that the needle-like and rhomboidal crystals had similar carotenoid composition and that they were composed mainly of ß-carotene accompanied by α-carotene. However, the presence of α-carotene was not identified in the helical crystals, which had the characteristic spatial structure. AFM measurements of crystals identified by Raman imaging revealed the crystal topography and showed the needle-like and rhomboidal crystals were planar but they differed in all three dimensions. Combining SNOM and Raman imaging enabled indication of carotenoid rich structures and visualised their distribution in the cell. The morphology of identified subcellular structures was characteristic for crystalline, membraneous and tubular chromoplasts that are plant organelles responsible for carotenoid accumulation in cells.