Assembly of high-quality genomes of the locoweed Oxytropis ochrocephala and its endophyte Alternaria oxytropis provides new evidence for their symbiotic relationship and swainsonine biosynthesis.

Locoweeds are perennial forbs poisonous to livestock and cause extreme losses to animal husbandry. Locoweed toxicity is attributed to the symbiotic endophytes in Alternaria sect. Undifilum, which produce a mycotoxin swainsonine (SW). We performed a de novo whole genome sequencing of the most common locoweed in China, Oxytropis ochrocephala (2n = 16), and assembled a high-quality, chromosome-level reference genome. Its genome size is 958.83 Mb with 930.94 Mb (97.09%) anchored and oriented onto eight chromosomes, and 31,700 protein-coding genes were annotated. Phylogenetic and collinearity analysis showed it is closely related to Medicago truncatula with a pair of large interchromosomal rearrangements, and both species underwent a whole-genome duplication event. We also derived the genome of A. oxytropis at 74.48 Mb with a contig N50 of 8.87 Mb and 10,657 protein-coding genes, and refined the genes of SW biosynthesis. Multiple Alternaria species containing the swnK gene were grouped into a single clade, but in other genera, swnK's homologues are diverse. Resequencing of 41 A. oxytropis strains revealed one SNP in the SWN cluster causing changes in SW concentration. Comparing the transcriptomes of symbiotic and nonsymbiotic interactions identified differentially expressed genes (DEGs) linked to defence and secondary metabolism in the host. Within the endophyte DEGs were linked to cell wall degradation, fatty acids and nitrogen metabolism. Symbiosis induced the upregulation of most of the SW biosynthetic genes. These two genomes and relevant sequencing data should provide valuable genetic resources for the study of the evolution, interaction, and SW biosynthesis in the symbiont.

The locoweed endophyte Alternaria oxytropis affects root development in Arabidopsis in vitro through auxin signaling and polar transport.

Locoweeds are leguminous forbs known for their toxicity to livestock caused by the endophytic fungi Alternaria sect. Undifilum. Unlike the defensive mutualisms reported in many toxin-producing endophytes and their plant hosts, the benefits that A. sect. Undifilum can confer to it host plants remains unclear. Here, we conducted physiological and genetic analyses to show that A. (sect. Undifilum) oxytropis influences growth, especially root development, in its locoweed host Oxytropis ochrocephala and Arabidopsis. The presence of A. oxytropis significantly decreased primary root length while increasing the numbers of lateral roots and root hairs, and increasing plant leaf area and fresh weight. The fungus also increased the concentrations of plant endogenous auxin, and the expression of key genes for auxin biosynthesis, signaling, and transport. These effects on root development were abolished in mutants deficient in auxin signaling and polar transport. Alternaria oxytropis down-regulated expression of PIN1 but increased expression of PIN2, PIN7, and AUX1, which might reflect alterations in the spatial accumulation of auxin responsible for the changes in root architecture. Plant growth was insensitive to A. oxytropis when naphthylphthalamic acid was applied. Our findings indicate a function of A. oxytropis in promoting the growth and development of Arabidopsis via the regulation of auxin, which in turn suggests a possible role in benefiting its locoweed hosts via a process independent of its toxin production.

Rethinking of the Roles of Endophyte Symbiosis and Mycotoxin in Oxytropis Plants.

Plants in the Oxytropis genus can live with the endophytic fungi Alternaria sect. Undifilum. Swainsonine, the mycotoxin produced by the endophyte render the host plant toxic and this has been detrimental to grazing livestock in both China and U.S.A. Despite previous efforts, many questions remain to be solved, such as the transmission mode and life cycle of host-endophyte symbiont, the biosynthesis pathway of swainsonine, and in particular the ecological role and evolution of such symbiosis. In this review, we compile the literature to synthesize ideas on the diversity of the symbiosis and propagation of the endophyte. We further compare the previous work from both Alternaria sect. Undifilum and other swainsonine producing fungi to orchestrate a more comprehensive biosynthesis pathway of swainsonine. We also connect swainsonine biosynthesis pathway with that of its precursor, lysine, and link this to a potential role in modulating plant stress response. Based on this we hypothesize that this host-endophyte co-evolution originated from the needs for host plant to adapt for stress. Validation of this hypothesis will depend on future research on endophytic symbiosis in Oxytropis and help in better understanding the roles of plant-endophyte symbiosis in non-Poaceae species.

ScPNP-A, a plant natriuretic peptide from Stellera chamaejasme, confers multiple stress tolerances in Arabidopsis.

As a class of peptide hormone, plant natriuretic peptides (PNPs) play an important role in maintaining water and salt balance in plants, as well as in the physiological processes of biotic stress and pathogen resistance. However, in plants, except for some PNPs, such as the Arabidopsis thaliana PNP-A (AtPNP-A), of which the function has not yet been thoroughly revealed, few PNPs in other plants have been reported. In this study, a PNP-A (ScPNP-A) has been identified and characterized in Stellera chamaejasme for the first time. ScPNP-A is a double-psi beta-barrel (DPBB) fold containing protein and is localized in the extracellular (secreted) space. In S. chamaejasme, the expression of ScPNP-A was significantly up-regulated by salt, drought and cold stress. Changes at the physiological and biochemical levels and the expression of resistance-related genes indicated that overexpression of ScPNP-A can significantly improve salt, drought and freezing tolerance in Arabidopsis. ScPNP-A could stimulate the opening, not the closing of stomata, and its expression was not enhanced by external application of ABA. Furthermore, overexpression of ScPNP-A resulted in the elevated expression of genes in the ABA biosynthesis and reception pathway. These suggested that there may be some cross-talk between ScPNP-A and the ABA-dependent signaling pathways to regulate water related stress, however further experimentation is required to understand this relationship. In addition, overexpression of ScPNP-A can enhance the resistance to pathogens by enhancing SAR in Arabidopsis. These results indicate that ScPNP-A could function as a positive regulator in plant response to biotic stress and abiotic stress.

OoNAC72, a NAC-Type Oxytropis ochrocephala Transcription Factor, Conferring Enhanced Drought and Salt Stress Tolerance in Arabidopsis.

The NAC proteins form one of the largest families of plant-specific transcription factors (TFs) and play essential roles in developmental processes and stress responses. In this study, we characterized a NAC domain transcription factor, OoNAC72, from a legume Oxytropis ochrocephala. OoNAC72 was proved to be localized in the nuclei in tobacco lower epidermal cells and had transcriptional activation activity in yeast, confirming its transcription activity. OoNAC72 expression could be induced by drought, salinity and exogenous abscisic acid (ABA) in O. ochrocephala seedlings. Furthermore, over-expression of OoNAC72 driven by CaMV35S promoter in Arabidopsis resulted in ABA hypersensitivity and enhanced tolerance to drought and salt stresses during seed germination and post-germinative growth periods. In addition, over-expression of OoNAC72 enhanced the expression of stress-responsive genes such as RD29A, RD29B, RD26, LEA14, ANACOR19, ZAT10, PP2CA, and NCED3. These results highlight the important regulatory role of OoNAC72 in multiple abiotic stress tolerance, and may provide an underlying reason for the spread of O. ochrocephala.

Two symmetrical unsaturated acids isolated from Viscum album.

In the present study, two new acetylene conjugate compounds, dibutyl (2Z, 6Z)-octa-2, 6-dien-4-yne dioate (1), and dibutyl (2E, 6E)- octa-2, 6-dien-4-yne dioate (2), were isolated from the dry stem leaves of Viscum album, along with nine known compounds (3 - 11). Their structures were confirmed on the basis of spectroscopic data. Compounds 1 and 8 showed antioxidant activity against xanthine oxidase (XOD) and 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydroxyl (DPPH), with the IC50 of 1.22 and 1.33 µmol·L-1, and the SC50 of 4.34 and 8.22 µmol·L-1, respectively.

Reference gene selection for qRT-PCR assays in Stellera chamaejasme subjected to abiotic stresses and hormone treatments based on transcriptome datasets.

BACKGROUND: Stellera chamaejasme Linn, an important poisonous plant of the China grassland, is toxic to humans and livestock. The rapid expansion of S. chamaejasme has greatly damaged the grassland ecology and, consequently, seriously endangered the development of animal husbandry. To draft efficient prevention and control measures, it has become more urgent to carry out research on its adaptive and expansion mechanisms in different unfavorable habitats at the genetic level. Quantitative real-time polymerase chain reaction (qRT-PCR) is a widely used technique for studying gene expression at the transcript level; however, qRT-PCR requires reference genes (RGs) as endogenous controls for data normalization and only through appropriate RG selection and qRT-PCR can we guarantee the reliability and robustness of expression studies and RNA-seq data analysis. Unfortunately, little research on the selection of RGs for gene expression data normalization in S. chamaejasme has been reported. METHOD: In this study, 10 candidate RGs namely, 18S, 60S, CYP, GAPCP1, GAPDH2, EF1B, MDH, SAND, TUA1, and TUA6, were singled out from the transcriptome database of S. chamaejasme, and their expression stability under three abiotic stresses (drought, cold, and salt) and three hormone treatments (abscisic acid, ABA; gibberellin, GA; ethephon, ETH) were estimated with the programs geNorm, NormFinder, and BestKeeper. RESULT: Our results showed that GAPCP1 and EF1B were the best combination for the three abiotic stresses, whereas TUA6 and SAND, TUA1 and CYP, GAPDH2 and 60S were the best choices for ABA, GA, and ETH treatment, respectively. Moreover, GAPCP1 and 60S were assessed to be the best combination for all samples, and 18S was the least stable RG for use as an internal control in all of the experimental subsets. The expression patterns of two target genes (P5CS2 and GI) further verified that the RGs that we selected were suitable for gene expression normalization. DISCUSSION: This work is the first attempt to comprehensively estimate the stability of RGs in S. chamaejasme. Our results provide suitable RGs for high-precision normalization in qRT-PCR analysis, thereby making it more convenient to analyze gene expression under these experimental conditions.