Complete chloroplast genomes of eight Delphinium taxa (Ranunculaceae) endemic to Xinjiang, China: insights into genome structure, comparative analysis, and phylogenetic relationships.

BACKGROUND: Delphinium L. represents a taxonomically intricate genus of significant phylogenetic and economic importance in Ranunculaceae. Despite the existence of few chloroplast genome datasets, a comprehensive understanding of genome structures and selective pressures within the genus remains unknown. Furthermore, several taxa in this genus are exclusively found in Xinjiang, China, a region renowned for its distribution and diversity of Chinese and Central Asian Delphinium species. Therefore, investigating the features of chloroplast genomes in this area will provide valuable insights into the evolutionary processes and phylogenetic relationships of the genus. RESULTS: In this study, the eight newly completed chloroplast genomes are examined, ranging in length from 153,979 bp to 154,284 bp. Alongside these, analysing six previously reported taxa re-annotated in Delphinium, 111 unique genes are identified across all samples. Genome structure, distributions of simple sequence repeats and short dispersed repeats, as well as gene content are similar among these Delphinium taxa. Nine hypervariable intergenic spacers and protein coding regions, including ndhF-trnL(TAG), rpl16-intron, rpl33, rps15, rps18, trnK(TTT)-trnQ(TTG), trnP(TGG)-psaJ, trnT(GGT)-psbD and ycf1, are identified among 13 perennial Delphinium. Selective pressure and codon usage bias of all the plastid genes are performed within 14 Delphinium taxa. Phylogenetic analysis based on 14 Delphinium plastomes, alongside two Aconitum (Ranunculaceae) species serving as outgroup taxa, reveals the monophyletic nature of Delphinium. Our findings further discern Delphinium into two distinct clades: perennial species (clade I) and annual species (clade II). In addition, compared with the nrDNA ITS topology, cytological data and morphological characters, D. mollifolium and D. maackianum showed potential involvement in hybridization or polyploidization processes. Excluding these two species, the perennial Delphinium (clade I) exhibits a stronger consistency with the morphology-based system that utilized seed morphology. CONCLUSION: This study represents the first comprehensive analysis of plastomic variations among Delphinium taxa, based on the examination of 14 complete plastomes. The chloroplast genome structure of Delphinium is similar to other angiosperms and possesses the typical quadripartite structure with the conserved genome arrangement and gene features. In addition, the variation of non-coding regions is larger than coding regions of the chloroplast genome. Through DNA sequence divergence across Delphinium plastomes and subsequent phylogenomic analyses ndhF-trnL(TAG) and ycf1 are identified as promising molecular markers. These highly variable loci held significant potential for future phylogenetic and phylogeographic studies on Delphinium. Our phylogenomic analyses based on the whole plastomes, concatenation of 132 unique intergenic spacer regions, concatenation of 77 unique protein-coding genes and nrDNA ITS, all support the monophyly of Delphinium and perennial taxa clusters together into one clade within this genus. These findings provide crucial data for systematic, phylogenomic and evolutionary research in the genus for future studies.

Male and female meiosis evince differential patterns in chiasma formation: a case study of ornamental plant, Delphinium ajacis L.

Chromosomal behaviour during megasporogenesis and microsporogenesis has been studied in ornamental Delphinium ajacis L. Meiosis in female sex cell initiates later than male. The floral buds which carry egg mother cell (EMC) at diplotene stage has pollen mother cells (PMCs) at tetrad stage of meiosis suggesting protandry. Although the 16 chromosomes formed regular eight bivalents in both the sex cells, they differed in overall chiasma frequency which was 32.95% higher in EMCs and found to be 18.52 ± 2.12 per cell. In PMCs, the average chiasma frequency recorded was 13.93 ± 1.40 per cell. Interestingly, this variation in chiasma frequency was largely confined to the two large bivalents which shared 42.61% chiasma per EMC. The use of Q-Q plot, Box plot and Whisker plot showed departure in the chiasma frequency distributions in EMCs and PMCs from the normal distribution pattern. The difference in chiasma frequency in the two sex cells was significant at all levels as indicated by the low P values of 3.094 × 10-11 obtained from nonparametric test, i.e. Wilcoxon rank-sum test. It is suggested that the two different mechanisms of recombination are operational in the two sex cells, and the sex differences of chiasma frequency could have arisen due to differential epigenetic modifications of the chromatin which pattern the double-strand breaks, and the position and frequency of crossing over visible as chiasmata.

Recurrent gene duplication in the angiosperm tribe Delphinieae (Ranunculaceae) inferred from intracellular gene transfer events and heteroplasmic mutations in the plastid matK gene.

The study of intracellular gene transfer may allow for the detection of interesting evolutionary processes such as ancient polyploidization. We compared 24 plastid genomes (plastomes) from tribe Delphinieae, one from tribe Nigelleae and one from tribe Ranunculeae, including five newly sequenced genomes. The functional transfers of the plastids rpl32 and rps16 to the nucleus in tribe Delphinieae were identified. Unexpectedly, we discovered multiple divergent copies of the nuclear-encoded plastid rpl32 in the genus Aconitum. Phylogenetic and synonymous substitution rate analyses revealed that the nuclear-encoded plastid rpl32 underwent two major duplication events. These ancient gene duplication events probably occurred via multiple polyploidization events in Aconitum between 11.9 and 24.7 Mya. Furthermore, our sequence rate analysis indicated that the eight plastid-encoded rpl subunits in Aconitum had a significantly accelerated evolutionary rate compared to those in other genera, suggesting that highly divergent paralogs targeted to the plastid may contribute to an elevated rate of evolution in plastid rpl genes. In addition, heteroplasmy of the plastid matK from two Aconitum species suggested the existence of potentially functional plastid maturases in its plastome. Our results provide insight into the evolutionary history of the tribe Delphinieae.

A double knockout mutant of acyl-glucose-dependent anthocyanin glucosyltransferase genes in Delphinium grandiflorum.

Blue coloration in delphinium flowers arises from 7-polyacylated anthocyanins which are modified alternately with acyl and glucosyl residues at the 7 position of the aglycone. Previously, we identified two independent genes for acyl-glucose-dependent anthocyanin 7-(6-(p-hydroxybenzoyl)-glucoside) glucosyltransferases (AA7BG-GT); recombinant proteins from the two cDNAs were produced in Escherichia coli and showed AA7BG-GT activity in vitro. Here, a double knockout mutant of both genes was found to lack modification of the second glucosyl residue following further acyl and glucosyl modifications. Both genes in the double mutant had nucleotide sequence changes and deletions that disrupted their transcripts and caused loss of AA7BG-GT activity in sepals. These results provide genetic confirmation that both genes are responsible for AA7BG-GT enzyme activity.

Identification of flavonoid 3'-hydroxylase in the yellow flower of Delphinium zalil.

The flowers of delphinium cultivars owe their coloration to anthocyanins such as delphinidin or pelargonidin derivatives. To date, no delphinium cultivars have been found with red flowers due to the presence of cyanidin derivatives. This suggests that delphiniums do not have cyanidin biosynthesis ability because of the loss of function of flavonoid 3' hydroxylase (F3'H). Here, we show that the wild delphinium species Delphinium zalil (synonym semibarbatum) can accumulate quercetin 3-glucosides in its sepals, presumably through F3'H activity. We isolated F3'H cDNA from D. zalil (DzF3'H) and produced a recombinant enzyme from a yeast transformant. The recombinant DzF3'H protein could convert naringenin, apigenin, dihydrokaempferol and kaempferol to eriodictyol, luteolin, dihydroquercetin and quercetin, respectively. An expression analysis confirmed that blue flowered D. grandiflorum does not express F3'H, and also showed that flavonoid 3',5'-hydroxylase and anthocyanidin synthase do not function in D. zalil sepals. DzF3'H can act as a flavonoid hydroxylase to produce cyanidin accumulation. The introduction of the DzF3'H gene into other delphinium species by conventional breeding may enable development of cultivars with novel flower colors.

Yeasts in nectar enhance male fitness in a montane perennial herb.

Floral nectar of many plant species is prone to colonization by microbial organisms such as yeasts. Their presence and metabolism of nectar chemical components have the potential to modify a suite of floral traits important for pollinator attraction, including nectar quality and scent. However, studies on the direct and indirect effects of nectar-inhabiting microorganisms on pollinator behavior and plant reproductive success remain rare. To determine their potential to affect pollinator behavior and plant fitness, we experimentally manipulated the common nectar-inhabiting yeast Metschnikowia reukaufii in the nectar of Delphinium nuttallianum, a short-lived montane perennial herb. We detected positive, indirect, pollinator-mediated effects of yeasts on male plant fitness measured as pollen donation using powdered fluorescent dyes. However, we detected no direct or indirect effects on components of female fitness. Matching effects on male plant fitness, pollinators responded positively to the presence of yeasts, removing more nectar from flowers treated with M. reukaufii. Our results provide evidence of effects of nectar-inhabiting yeasts on male plant fitness and highlight the importance of microorganisms in mediating plant-pollinator interactions and subsequent plant fitness.

Identification of the glucosyltransferase gene that supplies the p-hydroxybenzoyl-glucose for 7-polyacylation of anthocyanin in delphinium.

In delphiniums (Delphinium grandiflorum), blue flowers are produced by the presence of 7-polyacylated anthocyanins. The polyacyl moiety is composed of glucose and p-hydroxybenzoic acid (pHBA). The 7-polyacylation of anthocyanin has been shown to be catalysed by two different enzymes, a glucosyltransferase and an acyltransferase; both enzymes utilize p-hydroxybenzoyl-glucose (pHBG) as a bi-functional (Zwitter) donor. To date, however, the enzyme that synthesizes pHBG and the gene that encodes it have not been elucidated. Here, five delphinium cultivars were investigated and found to show reduced or undetectable 7-polyacylation activity; these cultivars synthesized delphinidin 3-O-rutinoside (Dp3R) to produce mauve sepals. One cultivar showed a deficiency for the acyl-glucose-dependent anthocyanin 7-O-glucosyltransferase (AA7GT) necessary for mediating the first step of 7-polyacylation. The other four cultivars showed both AA7GT activity and DgAA7GT expression; nevertheless, pHBG accumulation was significantly reduced compared with wild-type cultivars, whereas p-glucosyl-oxybenzoic acid (pGBA) was accumulated. Three candidate cDNAs encoding a UDP-glucose-dependent pHBA glucosyltransferase (pHBAGT) were identified. A phylogenetic analysis of DgpHBAGT amino acid sequences showed a close relationship with UGTs that act in acyl-glucose synthesis in other plant species. Recombinant DgpHBAGT protein synthesized pHBG and had a high preference for pHBA in vitro. Mutant cultivars accumulating pGBA had very low expression of DgpHBAGT, whereas expression during the development of sepals and tissues in a wild cultivar showed a close correlation to the level of accumulation of pHBG. These results support the conclusion that DgpHBAGT is responsible for in vivo synthesis of pHBG in delphiniums.

p-Hydroxybenzoyl-glucose is a zwitter donor for the biosynthesis of 7-polyacylated anthocyanin in Delphinium.

The blue color of delphinium (Delphinium grandiflorum) flowers is produced by two 7-polyacylated anthocyanins, violdelphin and cyanodelphin. Violdelphin is derived from the chromophore delphinidin that has been modified at the 7-position by Glc and p-hydroxybenzoic acid (pHBA) molecules. Modification of violdelphin by linear conjugation of Glc and pHBA molecules to a Glc moiety at the 7-position produces cyanodelphin. We recently showed that anthocyanin 7-O-glucosylation in delphinium is catalyzed by the acyl-Glc-dependent anthocyanin glucosyltransferase (AAGT). Here, we sought to answer the question of which enzyme activities are necessary for catalyzing the transfer of Glc and pHBA moieties to 7-glucosylated anthocyanin. We found that these transfers were catalyzed by enzymes that use p-hydroxybenzoyl-Glc (pHBG) as a bifunctional acyl and glucosyl donor. In addition, we determined that violdelphin is synthesized via step-by-step enzymatic reactions catalyzed by two enzymes that use pHBG as an acyl or glucosyl donor. We also isolated a cDNA encoding a protein that has the potential for p-hydroxybenzoylation activity and two AAGT cDNAs that encode a protein capable of adding Glc to delphinidin 3-O-rutinoside-7-O-(6-O-[p-hydroxybenzoyl]-glucoside) to form violdelphin.

A phylogeny of Delphinieae (Ranunculaceae) shows that Aconitum is nested within Delphinium and that Late Miocene transitions to long life cycles in the Himalayas and Southwest China coincide with bursts in diversification.

The tribe Delphinieae (Ranunculaceae) comprises two species-rich genera, Aconitum and Delphinium, the latter including Consolida and Aconitella. The 650-700 species are distributed in Eurasia and North America; three species occur on tropical African mountains. Maximum likelihood analyses of 2088 aligned nucleotides of plastid and nuclear sequences obtained from up to 185 species of Delphinieae from throughout the geographic range (plus relevant outgroups) show that three short-lived (facultative annual or biennial) Mediterranean species belonging to Delphinium subgenus Staphisagria are the sister clade to all other Delphinieae, implying that Staphisagria needs to be raised to genus status if Delphinium and Aconitum are to become mutually monophyletic. Molecular clock dating suggests an origin of the sampled Delphinieae in the Early Oligocene (c. 32.3 Ma) and expansion to North America of Aconitum and Delphinium around 3.3 and 2.9 Ma ago, respectively; the East African Mts. were reached by long-distance dispersal some 2.4 Ma ago, coincident with the major uplift of the East African Rift system. The ancestral growth form of the Delphinieae could not be reconstructed, but Late Miocene bursts in diversification rates in the Himalayan and southwestern Chinese clades of Aconitum and Delphinium appear to be associated with transitions from short-lived to long-lived life histories.

A novel glucosylation reaction on anthocyanins catalyzed by acyl-glucose-dependent glucosyltransferase in the petals of carnation and delphinium.

Glucosylation of anthocyanin in carnations (Dianthus caryophyllus) and delphiniums (Delphinium grandiflorum) involves novel sugar donors, aromatic acyl-glucoses, in a reaction catalyzed by the enzymes acyl-glucose-dependent anthocyanin 5(7)-O-glucosyltransferase (AA5GT and AA7GT). The AA5GT enzyme was purified from carnation petals, and cDNAs encoding carnation Dc AA5GT and the delphinium homolog Dg AA7GT were isolated. Recombinant Dc AA5GT and Dg AA7GT proteins showed AA5GT and AA7GT activities in vitro. Although expression of Dc AA5GT in developing carnation petals was highest at early stages, AA5GT activity and anthocyanin accumulation continued to increase during later stages. Neither Dc AA5GT expression nor AA5GT activity was observed in the petals of mutant carnations; these petals accumulated anthocyanin lacking the glucosyl moiety at the 5 position. Transient expression of Dc AA5GT in petal cells of mutant carnations is expected to result in the transfer of a glucose moiety to the 5 position of anthocyanin. The amino acid sequences of Dc AA5GT and Dg AA7GT showed high similarity to glycoside hydrolase family 1 proteins, which typically act as ß-glycosidases. A phylogenetic analysis of the amino acid sequences suggested that other plant species are likely to have similar acyl-glucose-dependent glucosyltransferases.

Establishment of zygomorphy on an ontogenic spiral and evolution of perianth in the tribe Delphinieae (Ranunculaceae).

BACKGROUND AND AIMS: Ranunculaceae presents both ancestral and derived floral traits for eudicots, and as such is of potential interest to understand key steps involved in the evolution of zygomorphy in eudicots. Zygomorphy evolved once in Ranunculaceae, in the speciose and derived tribe Delphinieae. This tribe consists of two genera (Aconitum and Delphinium s.l.) comprising more than one-quarter of the species of the family. In this paper, the establishment of zygomorphy during development was investigated to cast light on the origin and evolution of this morphological novelty. METHODS; The floral developmental sequence of six species of Ranunculaceae, three actinomorphic (Nigella damascena, Aquilegia alpina and Clematis recta) and three zygomorphic (Aconitum napellus, Delphinium staphisagria and D. grandiflorum), was compared. A developmental model was elaborated to break down the successive acquisitions of floral organ identities on the ontogenic spiral (all the species studied except Aquilegia have a spiral phyllotaxis), giving clues to understanding this complex morphogenesis from an evo-devo point of view. In addition, the evolution of symmetry in Ranunculaceae was examined in conjunction with other traits of flowers and with ecological factors. KEY RESULTS: In the species studied, zygomorphy is established after organogenesis is completed, and is late, compared with other zygomorphic eudicot species. Zygomorphy occurs in flowers characterized by a fixed merism and a partially reduced and transformed corolla. CONCLUSIONS: It is suggested that shifts in expression of genes controlling the merism, as well as floral symmetry and organ identity, have played a critical role in the evolution of zygomorphy in Delphinieae, while the presence of pollinators able to exploit the peculiar morphology of the flower has been a key factor for the maintenance and diversification of this trait.

Genetic diversity in Delphinium staphisagria (Ranunculaceae), a rare Mediterranean dysploid larkspur with medicinal uses.

Delphinium staphisagria is an endemic annual or biennial herb from the Mediterranean Basin, widely distributed in isolated populations of variable size. We evaluated the allozyme diversity of 31 populations along its distribution range via starch gel electrophoresis, assaying 12 enzyme systems and scoring 17 loci. The low levels of genetic variability detected (A = 11.8, A(p) = 1.6, H(o) = 0.026, H(e) = 0.057), are discussed in relation to the life-history traits of the species, such as short life-span, selfing or gravity seed dispersion. Other factors influencing genetic diversity, such as evolutionary history and spreading are also considered. Due to its historical medicinal uses, this plant has probably become widespread in the Mediterranean area. Human-mediated distribution could have promoted few migrant genotypes, recent founder events and long distance dispersal. These events would explain the genetic homogeneity found within and among populations, as well as the absence of a clear biogeographic structure. The limited genetic variability, the high genetic similarity among populations and the dysploidy of this species make it worthy of conservation. Management strategies are proposed mainly to preserve its genetic pool.

A QSAR toxicity study of a series of alkaloids with the lycoctonine skeleton.

A QSAR toxicity analysis has been performed for a series of 19 alkaloids with the lycoctonine skeleton. GA-MLRA (Genetic Algorithm combined with Multiple Linear Regression Analysis) technique was applied for the generation of two types of QSARs: first, models containing exclusively 3D-descriptors and second, models consisting of physicochemical descriptors. As expected, 3D-descriptor QSARs have better statistical fits. Physicochemical-descriptor containing models, that are in a good agreement with the mode of toxic action exerted by the alkaloids studied, have also been identified and discussed. In particular, TPSA (Topological Polar Surface Area) and nC=O (number of -C(O)- fragments) parameters give the best statistically significant mono- and bidescriptor models (when combined with lipophilicity, MlogP) confirming the importance of H-bonding capability of the alkaloids for binding at the receptor site.