Formation of the Fungal Indole Alkaloid Speradine F Implies Multiple Nonenzymatic Oxidation Steps.

The highly oxygenated indole alkaloid speradine F (4) with a 6/5/6/5/5/5 hexacyclic skeleton was isolated from a culture of Penicillium palitans, together with its precursors ß-cyclopiazonic acid (ß-CPA, 5) and cyclopiazonic acid (CPA, 1). Gene deletion and heterologous expression led to the identification of the responsible five-gene spe cluster for the speradine skeleton formation. Precursor supply experiments proved that 1 was enzymatically converted, via 2-oxoCPA (2), to speradine A (3), which subsequently undergoes multistep nonenzymatic hydroxylations to 4.

Biosynthesis of Viridicatol in Penicillium palitans Implies a Cytochrome P450-Mediated meta Hydroxylation at a Monoalkylated Benzene Ring.

Cyclopenol (1) and viridicatol (6) with m-hydroxyl groups were isolated from a culture of Penicillium palitans. Genome mining and heterologous expression in Aspergillus nidulans led to the identification of their biosynthetic gene cluster and the cytochrome P450 enzyme VdoD responsible for the meta hydroxylation. Precursor feeding experiments into vdoD transformant proved the conversion of cyclopenin (2) to 1, which then undergoes a spontaneous or VdoA-catalyzed rearrangement to 6. A direct conversion of viridicatin (5) to 6 by VdoD was not detected.

Chromosome-Scale Genome Assembly of the Resurrection Plant Acanthochlamys bracteata (Velloziaceae).

Acanthochlamys bracteata (Velloziaceae) is a resurrection plant with cold tolerance. Herein, a chromosome-level reference genome of A. bracteata based on Nanopore, Illumina, and Hi-C data is reported. The high-quality assembled genome was 197.97 Mb, with a scaffold N50 value of 8.64 Mb and a contig N50 value of 6.96 Mb. We annotated 23,509 protein-coding genes. Eight contracted gene families and three expanded gene families were detected. Repeat sequences accounted for approximately 28.63% of the genome. The LEA1 and Dehydrin gene families, which are involved in desiccation resistance, expanded in A. bracteata. We identified genes involved in chilling tolerance, COLD1.

HAG1 and SWI3A/B control of male germ line development in P. patens suggests conservation of epigenetic reproductive control across land plants.

KEY MESSAGE: Bryophytes as models to study the male germ line: loss-of-function mutants of epigenetic regulators HAG1 and SWI3a/b demonstrate conserved function in sexual reproduction. With the water-to-land transition, land plants evolved a peculiar haplodiplontic life cycle in which both the haploid gametophyte and the diploid sporophyte are multicellular. The switch between these phases was coined alternation of generations. Several key regulators that control the bauplan of either generation are already known. Analyses of such regulators in flowering plants are difficult due to the highly reduced gametophytic generation, and the fact that loss of function of such genes often is embryo lethal in homozygous plants. Here we set out to determine gene function and conservation via studies in bryophytes. Bryophytes are sister to vascular plants and hence allow evolutionary inferences. Moreover, embryo lethal mutants can be grown and vegetatively propagated due to the dominance of the bryophyte gametophytic generation. We determined candidates by selecting single copy orthologs that are involved in transcriptional control, and of which flowering plant mutants show defects during sexual reproduction, with a focus on the under-studied male germ line. We selected two orthologs, SWI3a/b and HAG1, and analyzed loss-of-function mutants in the moss P. patens. In both mutants, due to lack of fertile spermatozoids, fertilization and hence the switch to the diploid generation do not occur. Pphag1 additionally shows arrested male and impaired female gametangia development. We analyzed HAG1 in the dioecious liverwort M. polymorpha and found that in Mphag1 the development of gametangiophores is impaired. Taken together, we find that involvement of both regulators in sexual reproduction is conserved since the earliest divergence of land plants.

Plastome of the mycoheterotrophic eudicot Exacum paucisquama (Gentianaceae) exhibits extensive gene loss and a highly expanded inverted repeat region.

Mycoheterotrophic plants are highly specialized species able to acquire organic carbon from symbiotic fungi, with relaxed dependence on photosynthesis for carbon fixation. The relaxation of the functional constraint of photosynthesis and thereby the relaxed selective pressure on functional photosynthetic genes usually lead to substantial gene loss and a highly degraded plastid genome in heterotrophs. In this study, we sequenced and analyzed the plastome of the eudicot Exacum paucisquama, providing the first plastid genome of a mycoheterotroph in the family Gentianaceae to date. The E. paucisquama plastome was 44,028 bp in length, which is much smaller than the plastomes of autotrophic eudicots. Although the E. paucisquama plastome had a quadripartite structure, a distinct boundary shift was observed in comparison with the plastomes of other eudicots. We detected extensive gene loss and only 21 putative functional genes (15 protein-coding genes, four rRNA genes and two tRNA genes). Our results provide valuable information for comparative evolutionary analyses of plastomes of heterotrophic species belonging to different phylogenetic groups.

Plastid Genome Evolution in the Subtribe Calypsoinae (Epidendroideae, Orchidaceae).

Calypsoinae is a small subtribe in Orchidaceae (Epidendroideae) characterized by diverse trophic strategies and morphological characters. Calypsoinae includes 13 genera, four of which are leafless and mycoheterotrophic. Mycoheterotrophic species in the leafless genus Corallorhiza are well suited to studies of plastome evolution. However, the lack of plastome sequences for other genera in Calypsoinae limits the scope of comparative and phylogenetic analyses, in particular our understanding of plastome evolution. To understand plastid genome evolution in Calypsoinae, we newly sequenced the plastomes of 12 species in the subtribe, including representatives of three mycoheterotrophic genera as well as five autotrophic genera. We detected two parallel photosynthetic losses in Corallorhiza. Evolutionary analyses indicated that the transition to obligate mycoheterotrophy leads to the relaxation of selection in a highly gene-specific pattern.

Phylogenomics of Orchidaceae based on plastid and mitochondrial genomes.

To advance our knowledge of orchid relationships and timing of their relative divergence, we used 76 protein-coding genes from plastomes (ptCDS) and 38 protein-coding genes from mitochondrial genomes (mtCDS) of 74 orchids representing the five subfamilies and 18 tribes of Orchidaceae, to reconstruct the phylogeny and temporal evolution of the Orchidaceae. In our results, the backbone of orchid tree well supported with both datasets, but there are conflicts between these trees. The phylogenetic positions of two subfamilies (Vanilloideae and Cypripedioideae) are reversed in these two analyses. The phylogenetic positions of several tribes and subtribes, such as Epipogiinae, Gastrodieae, Nerviliinae, and Tropidieae, are well resolved in mtCDS tree. Thaieae have a different position among higher Epidendroideae, instead of sister to the higher Epidendroideae. Interrelationships of several recently radiated tribes within Epidendroideae, including Vandeae, Collabieae, Cymbidieae, Epidendreae, Podochileae, and Vandeae, have good support in the ptCDS tree, but most are not resolved in the mtCDS tree. Conflicts between the two datasets may be attributed to the different substitution rates in these two genomes and heterogeneity of substitution rate of plastome. Molecular dating indicated that the first three subfamilies, Apostasioideae, Cypripedioideae and Vanilloideae, diverged relatively quickly, and then there was a longer period before the last two subfamilies, Orchidoideae and Epidendroideae, began to radiate. Most mycoheterotrophic clades of Orchidaceae evolved in the last 30 million years with the exception of Gastrodieae.

Evolution of plastid genomes of Holcoglossum (Orchidaceae) with recent radiation.

BACKGROUND: The plastid is a semiautonomous organelle with its own genome. Plastid genomes have been widely used as models for studying phylogeny, speciation and adaptive evolution. However, most studies focus on comparisons of plastid genome evolution at high taxonomic levels, and comparative studies of the process of plastome evolution at the infrageneric or intraspecific level remain elusive. Holcoglossum is a small genus of Orchidaceae, consisting of approximately 20 species of recent radiation. This made it an ideal group to explore the plastome mutation mode at the infrageneric or intraspecific level. RESULTS: In this paper, we reported 15 complete plastid genomes from 12 species of Holcoglossum and 1 species of Vanda. The plastid genomes of Holcoglossum have a total length range between 145 kb and 148 kb, encoding a set of 102 genes. The whole set of ndh-gene families in Holcoglossum have been truncated or pseudogenized. Hairpin inversion in the coding region of the plastid gene ycf2 has been found. CONCLUSIONS: Using a comprehensive comparative plastome analysis, we found that all the indels between different individuals of the same species resulted from the copy number variation of the short repeat sequence, which may be caused by replication slippage. Annotation of tandem repeats shows that the variation introduced by tandem repeats is widespread in plastid genomes. The hairpin inversion found in the plastid gene ycf2 occurred randomly in the Orchidaceae.

[Spatial variability and management zone of soil major nutrients in tobacco fields in Qiannan mountainous region].

Spatial variability and management zone of soil major nutrients in tobacco fields in Qian-nan mountainous region were analyzed using geostatistics and fuzzy c-mean algorithm. Results indicated that the level of soil organic matter (OM) was moderate, and alkalytic nitrogen (AN), available phosphorus (AP) and available potassium (AK) were rich according to tobacco soil nutrient classification standards. Coefficients of variation (CV) of OM, AN, AP and AK were moderate. Contents of OM, AN, AP and AK fitted log-normal distributions. Correlation analysis showed moderate correlations between OM and AN, AP and AK. OM and AN were best described by Gaussian semivariogram models, while AP and AK were described by exponential models. The four nutrients displayed moderate spatial autocorrelation. There were significant differences among lag distances of four soil nutrients. OM, AN, AP and AK in the majority of studied regions varied at moderate to very rich levels, and deficiencies of OM, AN, AP and AK only accounted for 0.93%, 0.53%, 0.24% and 7.91% of the total studied region, respectively. Based on the results, the studied region was divided into two management zones (MZ), namely MZ1 and MZ2, accounting for 69. 8% and 30. 2% of the studied region respectively. The soil levels of OM, AN, AP and AK in MZ1 were significantly lower than those in MZ2 (P < 0.01).

Separation and characterization of sucrose esters from Oriental tobacco leaves using accelerated solvent extraction followed by SPE coupled to HPLC with ion-trap MS detection.

Sucrose esters (SEs) were successfully extracted from Oriental tobacco leaves using a new methodology based on accelerated solvent extraction followed by hydrophilic-lipophilic balanced cartridge cleanup step. The SEs were detected by HPLC with ion-trap MS detection using an electrospray interface operated in the positive ion mode. This methodology combines the high efficiency of extraction provided by a pressurized fluid and the highly sensitive characterization offered by ion-trap MS. Under the optimized conditions, 14 SEs were first identified among a total of 23 SEs found in Oriental tobacco leaves. Under the same conditions, only four new SEs were extracted by using traditional ultrasound-assisted extraction and liquid-solid extraction methods. The present method might be potentially useful in high-efficiency extraction and sensitive characterization of SEs from complex matrices such as tobacco leaves.