Chloroplast genome analysis and evolutionary insights in the versatile medicinal plant Calendula officinalis L.

Calendula officinalis L.is a versatile medicinal plant with numerous applications in various fields. However, its chloroplast genome structure, features, phylogeny, and patterns of evolution and mutation remain largely unexplored. This study examines the chloroplast genome, phylogeny, codon usage bias, and divergence time of C. officinalis, enhancing our understanding of its evolution and adaptation. The chloroplast genome of C. officinalis is a 150,465 bp circular molecule with a G + C content of 37.75% and comprises 131 genes. Phylogenetic analysis revealed a close relationship between C. officinalis, C. arvensis, and Osteospermum ecklonis. A key finding is the similarity in codon usage bias among these species, which, coupled with the divergence time analysis, supports their close phylogenetic proximity. This similarity in codon preference and divergence times underscores a parallel evolutionary adaptation journey for these species, highlighting the intricate interplay between genetic evolution and environmental adaptation in the Asteraceae family. Moreover unique evolutionary features in C. officinalis, possibly associated with certain genes were identified, laying a foundation for future research into the genetic diversity and medicinal value of C. officinalis.

Heterologous Production of Calendic Acid Naturally Found in Calendula officinalis by Recombinant Fission Yeast.

Calendic acid (CA) is a conjugated fatty acid with anti-cancer properties that is widely present in seed oil of Calendula officinalis. Using the co-expression of C. officinalis fatty acid conjugases (CoFADX-1 or CoFADX-2) and Punica granatum fatty acid desaturase (PgFAD2), we metabolically engineered the synthesis of CA in the yeast Schizosaccharomyces pombe without the need for linoleic acid (LA) supplementation. The highest CA titer and achieved accumulation were 4.4 mg/L and 3.7 mg/g of DCW in PgFAD2 + CoFADX-2 recombinant strain cultivated at 16 °C for 72 h, respectively. Further analyses revealed the accumulation of CA in free fatty acids (FFA) and downregulation of the lcf1 gene encoding long-chain fatty acyl-CoA synthetase. The developed recombinant yeast system represents an important tool for the future identification of the essential components of the channeling machinery to produce CA as a high-value conjugated fatty acid at an industrial level.

DNA barcoding and phylogenetic analysis of leafhoppers associated with Aster Yellow disease on China aster, Marigold and Chrysanthemum.

The Cicadellidae (Auchenorrhyncha: Hemiptera) are important agricultural, horticultural and ornamental pests. But it is very difficult to define nymphs and female adults using morphological characteristics. This research was aimed at understanding the variety of leafhoppers species and defining the prospective cause of the aster-yellow disease in China Aster, Marigold and Chrysanthemum. Two surveys were conducted in and around Pune, Maharashtra and Bengaluru, Karnataka between November 2016 and February 2017. The mitochondrial cytochrome oxidase subunit I (mtCOI) region marker was used in the species diagnosis and genetic diversity research. Through the use of mtCOI molecular marker eight different leafhoppers species were identified as Sogatella furcifera, Homalodisca insolita, Amrasca biguttula, Balclutha incise and Balclutha abdominalis and Japanagallia trifurcate. Whereas at genus level identified as Toya, Empoasca, Perkinsiella, Hishimonus, Tambocerus, Phaconeura, Curena, Psammotettix and Graphocophala species. These results are strongly corroborated with morphological identification. On the basis of multiple sequence alignment of the mtCOI gene, a species phylogenetic tree with the highest likelihood was drawn. All the leafhopper species clustered together in accordance with the species data collected from the database of the different geographic regions from the NCBI GenBank and Barcode of Life (BOLD). Such results suggest that it is important to use both molecular and morphological methods to ensure accurate identification of organisms. To conclude, this research contributes valuable knowledge to molecular biology and recognizes leafhopper species that serve as major phytoplasma vectors.

Phenotypic and molecular cytogenetic variability in calendula (Calendula officinalis L.) cultivars and mutant lines obtained via chemical mutagenesis.

The morphological, meiotic and chromosomal variability were studied in two cultivars of Calendula officinalis L. and their mutant lines obtained though chemical mutagenesis using diethyl sulphate (DES) (0.04%, 0.08%) and dimethyl sulphate (DMS) (0.025%, 0.05%). The studied cultivars displayed different sensitivity to DMS and DES mutagens. More M1 plants with morphological changes were observed in C. officinalis cv. 'Zolotoe more' than in cv. 'Rajskij sad'. DMS and DES at low concentrations had positive effects on main agro-metrical traits in both cultivars including plant height, inflorescence diameter and number of inflorescences per plant. Dose-dependent increase in number of various meiotic abnormalities was revealed in both mutant lines. Comparative karyotype analysis and FISH-based visualization of 45S and 5S rDNA indicated a high level of karyotype stability in M1 and M2 plants. Seed treatments with DMS and DES at certain concentrations resulted in higher yields of inflorescences in M1 plants compared to the control. In M2 generation, dose-dependent reduction in the yields of inflorescences was observed. Our findings demonstrate that DMS and DES at low concentrations have great potential in calendula mutation breeding.

Mutagenic influences of colchicine on phenological and molecular diversity of Calendula officinalis L.

Six different colchicine concentrations: 0, 400, 800, 1200, 1600, and 2000 ppm, in combination with four soaking time treatments (1, 2, 3, and 4 h), were selected to assess the effects on germination, vegetative growth, and flower yield components in calendula plants. The molecular diversity among the treatments was assessed using ten SRAP marker combinations. Seed soaking in colchicine significantly enhanced both the fresh and the dry shoot and root masses, flowering date, number of flowers per plant, and flower diameter. At 1200-ppm colchicine combined with a 4-h soaking time, a superior effect on seed germination was observed, whereas 800 ppm for 4 h produced the highest number of flowers and the largest flower diameter. The earliest flowering time was found at 800 ppm combined with a short soaking time (1 h), while the 4-h soaking time with 800 ppm, is recommended for growing calendula outdoors, since it enhances flower development. At the molecular level, 752 fragments were successfully amplified using the SRAP primers, with 280 genetic loci found throughout the calendula genome. The polymorphism percentage ranged from 79 to 100% and the polymorphic information content (PIC) values ranged between 0.85 and 0.97. The high number of detected loci and PIC values suggests a great power of SRAP markers in detecting mutant molecular diversity. Our results clearly show the existence of genetic variation among colchicine treated calendula plants and the clustering of the studied mutants was concordant with the colchicine concentration used.

Phenotypic and biochemical profile changes in calendula (Calendula officinalis L.) plants treated with two chemical mutagenesis.

Chemical mutagenesis is an efficient tool used in mutation-breeding programs to improve the vital characters of the floricultural crops. This study aimed to estimate the effects of different concentrations of two chemical mutagens; sodium azide (SA) and diethyl sulfate (DES). The vegetative growth and flowering characteristics in two generations (M1 and M2) of calendula plants were investigated. Seeds were treated with five different concentrations of SA and DES (at the same rates) of 1000, 2000, 3000, 4000, and 5000 ppm, in addition to a control treatment of 0 ppm. Results showed that lower concentrations of SA mutagen had significant effects on seed germination percentage, plant height, leaf area, plant fresh weight, flowering date, inflorescence diameter, and gas-exchange measurements in plants of both generations. Calendula plants tended to flower earlier under low mutagen concentrations (1000 ppm), whereas higher concentrations delayed flowering significantly. Positive results on seed germination, plant height, number of branches, plant fresh weight, and leaf area were observed in the M2-generation at lower concentrations of SA (1000 ppm), as well as at 4000 ppm DES on number of leaves and inflorescences. The highest total soluble protein was detected at the concentrations of 1000 ppm SA and 2000 ppm DES. DES showed higher average of acid phosphatase activity than SA. Results indicated that lower concentrations of SA and DES mutagens had positive effects on seed germination percentage, plant height, leaf area, plant fresh weight, flowering date, inflorescence diameter, and gas-exchange measurements. Thus, lower mutagen concentrations could be recommended for better floral and physio-chemical performance.

Production of oleanolic acid glycosides by hairy root established cultures of Calendula officinalis L.

In order to initiate hairy root culture initiation cotyledons and hypocotyls of Calendula officinalis L. were infected with Agrobacterium rhizogenes strain ATCC 15834 or the same strain containing pCAMBIA 1381Z vector with ß-glucuronidase reporter gene under control of promoter of NIK (Nematode Induced Kinase) gene. The efficiency of induction of hairy roots reached 33.8% for cotyledons and 66.6% for hypocotyls together for both transformation experiments. Finally, eight control and nine modified lines were established as a long-term culture. The hairy root cultures showed the ability to synthesize oleanolic acid mainly (97%) as glycosides; control lines contained it at the average 8.42 mg · g(-1) dry weight in tissue and 0.23 mg · dm(-3) in medium; modified lines: 4.59 mg · g(-1) for the tissue, and 0.48 mg · dm(-3) for the medium. Additionally lines showed high positive correlation between dry/fresh weight and oleanolic acid concentration in tissue. Using the Killiani mixture in acidic hydrolysis of oleanolic acid glycosides released free aglycones that were partially acetylated in such conditions.

Carotenoid isomerase is key determinant of petal color of Calendula officinalis.

Orange petals of calendula (Calendula officinalis) accumulate red carotenoids with the cis-configuration at the C-5 or C-5' position (5-cis-carotenoids). We speculated that the orange-flowered calendula is a carotenoid isomerase (crtiso) loss-of-function mutant that impairs the cis-to-trans conversion of 5-cis-carotenoids. We compared the sequences and enzyme activities of CRTISO from orange- and yellow-flowered calendulas. Four types of CRTISO were expressed in calendula petals. The deduced amino acid sequence of one of these genes (CoCRTISO1) was different between orange- and yellow-flowered calendulas, whereas the sequences of the other three CRTISOs were identical between these plants. Analysis of the enzymatic activities of the CoCRTISO homologs showed that CoCRTISO1-Y, which was expressed in yellow petals, converted carotenoids from the cis-to-trans-configuration, whereas both CoCRTISO1-ORa and 1-ORb, which were expressed in orange petals, showed no activity with any of the cis-carotenoids we tested. Moreover, the CoCRTISO1 genotypes of the F2 progeny obtained by crossing orange and yellow lines linked closely to petal color. These data indicate that CoCRTISO1 is a key regulator of the accumulation of 5-cis-carotenoids in calendula petals. Site-directed mutagenesis showed that the deletion of Cys-His-His at positions 462-464 in CoCRTISO1-ORa and a Gly-to-Glu amino acid substitution at position 450 in CoCRTISO1-ORb abolished enzyme activity completely, indicating that these amino acid residues are important for the enzymatic activity of CRTISO.

Identification and analysis of a gene from Calendula officinalis encoding a fatty acid conjugase.

Two homologous cDNAs, CoFad2 and CoFac2, were isolated from a Calendula officinalis developing seed by a polymerase chain reaction-based cloning strategy. Both sequences share similarity to FAD2 desaturases and FAD2-related enzymes. In C. officinalis plants CoFad2 was expressed in all tissues tested, whereas CoFac2 expression was specific to developing seeds. Expression of CoFad2 cDNA in yeast (Saccharomyces cerevisiae) indicated it encodes a Delta12 desaturase that introduces a double bond at the 12 position of 16:1(9Z) and 18:1(9Z). Expression of CoFac2 in yeast revealed that the encoded enzyme acts as a fatty acid conjugase converting 18:2(9Z, 12Z) to calendic acid 18:3(8E, 10E, 12Z). The enzyme also has weak activity on the mono-unsaturates 16:1(9Z) and 18:1(9Z) producing compounds with the properties of 8,10 conjugated dienes.

Formation of conjugated delta8,delta10-double bonds by delta12-oleic-acid desaturase-related enzymes: biosynthetic origin of calendic acid.

Divergent forms of the plant Delta(12)-oleic-acid desaturase (FAD2) have previously been shown to catalyze the formation of acetylenic bonds, epoxy groups, and conjugated Delta(11),Delta(13)-double bonds by modification of an existing Delta(12)-double bond in C(18) fatty acids. Here, we report a class of FAD2-related enzymes that modifies a Delta(9)-double bond to produce the conjugated trans-Delta(8),trans-Delta(10)-double bonds found in calendic acid (18:3Delta(8trans,10trans,12cis)), the major component of the seed oil of Calendula officinalis. Using an expressed sequence tag approach, cDNAs for two closely related FAD2-like enzymes, designated CoFADX-1 and CoFADX-2, were identified from a C. officinalis developing seed cDNA library. The deduced amino acid sequences of these polypeptides share 40-50% identity with those of other FAD2 and FAD2-related enzymes. Expression of either CoFADX-1 or CoFADX-2 in somatic soybean embryos resulted in the production of calendic acid. In embryos expressing CoFADX-2, calendic acid accumulated to as high as 22% (w/w) of the total fatty acids. In addition, expression of CoFADX-1 and CoFADX-2 in Saccharomyces cerevisiae was accompanied by calendic acid accumulation when induced cells were supplied exogenous linoleic acid (18:2Delta(9cis,12cis)). These results are thus consistent with a route of calendic acid synthesis involving modification of the Delta(9)-double bond of linoleic acid. Regiospecificity for Delta(9)-double bonds is unprecedented among FAD2-related enzymes and further expands the functional diversity found in this family of enzymes.