Phenotypic Plasticity and Local Adaptation of Leaf Cuticular Waxes Favor Perennial Alpine Herbs under Climate Change

Six perennial herbs (Plantago asiatica, Polygonum viviparum, Anaphalis lactea, Kobresia humilis, Leontopodium nanum and Potentilla chinensis) widely distributed in alpine meadows were reciprocally transplanted at two sites in eastern edge of Qinghai-Tibetan Plateau, Hongyuan (3434 m, 2.97 °C, 911 mm) and Qilian (3701 m, 2.52 °C, 472 mm), aiming to evaluate the responses of alpine plants to changing environments. When plants were transplanted from Hongyuan to Qilian, most plant species showed a decrease of total wax coverage in first year and reverse trend was observed for some plant species in second year. However, when plants were transplanted from Qilian to Hongyuan, the response of total wax coverage differed greatly between plant species. When compared with those in first year, plasticity index of average chain length of alkane decreased whereas carbon preference index of alkane increased at both Hongyuan and Qilian in second year. The total wax coverage differed between local and transplanted plants, suggesting both environmental and genetic factors controlled the wax depositions. Structural equation modeling indicated that co-variations existed between leaf cuticular waxes and leaf functional traits. These results suggest that alpine herbs adjust both wax depositions and chain length distributions to adapt to changing environment, showing climate adaptations.

Application of Multiplex TaqMan Real-Time PCR Assay in Survey of Five Lily Viruses Infecting Lilium spp.

Lily symptomless virus (LSV), Lily mottle virus (LMoV), Cucumber mosaic virus (CMV), Shallot yellow stripe virus (SYSV), and Plantago asiatica mosaic virus (PlAMV) are five of the economically important viruses infecting lilies (Lilium spp.) worldwide. In order to prevent the occurrence and spread of these viruses, it is necessary to develop a rapid, effective, and sensitive detection method for the simultaneous detection and specific quantification of these viruses. In this study, specific primers and probes for multiplex TaqMan real-time PCR assays designed from conserved regions of the coat protein sequence of each virus were used for the simultaneous detection of these viruses in lilies (Lilium spp.). The optimal concentration of primers and probes and reaction annealing temperature were 20 µM and 55.9 °C, respectively. The detection limits of the assay were 1.33 × 102, 1.27 × 101, 1.28 × 101, 2.33 × 102, and 2.01 × 102 copies·μL−1 for LSV, LMoV, CMV, SYSV, and PlAMV, respectively. Specificity was determined using seven viral pathogens of lilies. Variability tests of intra- and inter-assays showed high reproducibility with coefficients of variation <2%. The multiplex TaqMan real-time PCR assay was used to detect these viruses from lily samples in China. In brief, our developed assay showed high specificity, sensitivity, and reproducibility for the simultaneous detection and differentiation of five lily-infecting viruses and can be used for certification and quarantine programs.

Phytoremediation Potential of Four Native Plants in Soils Contaminated with Lead in a Mining Area

Metal pollution in soils is an issue of global concern, and lead (Pb) pollution is considered to be the most serious type. The arid and semi-arid areas of Northwest China are rich in Pb ore resources. In this study, four native crops [wormwood (Artemisia capillaris), dandelion (Taraxacum mongolicum), alfalfa (Medicago sativa), and plantain (Plantago asiatica L.)] that grow naturally around tailings slag in a mining area in Northwest China were selected to screen their ecological restoration impacts on Pb-contaminated soil. In pot experiments, four different metal lead pollution gradients (0, 2, 3, and 5‰ w/w) were set, and crop growth indexes such as plant height, root length, and biomass, together with the changes of soil Pb content in different tissues and organs before and after planting were analyzed. The results showed the difference in the amount of accumulated Pb in relation to the level of Pb content in the soil. It was found that wormwood and plantain have great potential as remediation plants for soil metal lead pollution in the arid and semi-arid lead bearing mining areas of Northwest China.

First report of bean common mosaic virus infecting heavenly bamboo (Nandina domestica) in China

Heavenly bamboo (Nandina domestica) is an evergreen ornamental plant with worldwide distribution. In May 2018, seven out of twenty N. domestica plants showing virus-like symptoms, such as yellow mosaic and curling, were observed in Lin’an, Zhejiang province. To determine the causal agent, a small RNA library was constructed using the Small RNA v1.5 Sample Prep Kit (Illumina, San Diego, USA) with total RNA extracted from leaves of a symptomatic plant. The library was sequenced by the Solexa platform at BGI Genomics (Shenzhen, China). A total number of 21,071,675 high-quality reads of 17-28 nucleotides (nt) in length remained after trimming adapter sequences and quality control. Reads were assembled using Velvet 0.7.31 and Oases 0.2.07 with the k-mer value of 17 (Schulz et al. 2012). BlastN and BlastX search against the GenBank viral nonredundant sequence databases revealed fifty-six contigs homologous to bean common mosaic virus (BCMV; genus Potyvirus; family Potyviridae). No contig homologous to the genomic sequence of other plant-infecting viruses was identified. These contigs were further assembled into a 9,315-nt fragment by SeqMan Pro 7.1.0 in Lasergene package (DNASTAR, Madison, WI), which covered 92.68% of the genome of BCMV strain CT (BCMV-CT; GenBank accession no. KM076650). The genome of this BCMV isolate (BCMV-NTZ1) was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using primers designed based on assembled contigs with the Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Beijing, China) and the FirstChoice® RLM-RACE Kit (Invitrogen, Carlsbad, USA), respectively. Amplicons were cloned and Sanger sequenced with three independent clones per amplicon. The genome is 10,052 nt in length excluding the poly-A tail (Genbank accession no. MZ670770) and shared the highest nt sequence identities with BCMV-CT (88.46%). The putative polyprotein shared 93.36% amino acid (aa) sequence identity with that of BCMV-CT. BCMV-NTZ1 also clustered with BCMV-CT in phylogenetic trees based on BCMV full genomes and aa sequences of coat protein. Five-leaf-stage seedlings of Nicotiana tabacum, N. benthamiana, Glycine max (Linn.) Merr., and Capsicum frutescens were mechanically inoculated with sap of BCMV-infected N. domestica leaves at fifteen plants per species. Seedlings of G. max developed virus-like (mosaic and leaf deformity) symptoms (7/15) at 15 days post-inoculation, while other plants remained symptomless throughout the experiment. Subsequent RT-PCR on all the plants using primers 27F1/14Rter and sequencing confirmed the presence and absence of BCMV-NTZ1 in all symptomatic G. max seedlings and other asymptomatic indicator plants, respectively. Subsequent RT-PCR survey further confirmed the association of BCMV with symptomatic heavenly bamboo samples but not asymptomatic plants (7/20). To the best of our knowledge, this is the first report of BCMV naturally infecting heavenly bamboo in China. N. domestica is susceptible to many viruses, e.g., cucumber mosaic virus, plantago asiatica mosaic virus, nandina stem pitting virus, apple stem grooving virus, and alternanthera mosaic virus (Barnett et al. 1973; Ahmed et al. 1983; Hughes et al. 2002, 2005; Tang et al. 2010; Wei et al. 2015). Our results indicate that N. domestica can also serve as an overwinter reservoir for BCMV and special attention should be paid to the damage it may cause.

A Polysaccharide From the Whole Plant of Plantago asiatica L. Enhances the Antitumor Activity of Dendritic Cell-Based Immunotherapy Against Breast Cancer

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs) that mediate T-cell immune responses. Breast cancer is one of the most commonly diagnosed diseases and its mortality rate is higher than any other cancer in both humans and canines. Plantain polysaccharide (PLP), extracted from the whole plant of Plantago asiatica L., could promote the maturation of DCs. In this research, we found that PLP could upregulate the maturation of DCs both in vitro and in vivo. PLP-activated DCs could stimulate lymphocytes’ proliferation and differentiate naive T cells into cytotoxic T cells. Tumor antigen-specific lymphocyte responses were enhanced by PLP and CIPp canine breast tumor cells lysate-pulsed DCs, and PLP and CIPp-cell-lysate jointly stimulated DCs cocultured with lymphocytes having the great cytotoxicity on CIPp cells. In the 4T1 murine breast tumor model, PLP could control the size of breast tumors and improve immunity by recruiting DCs, macrophages, and CD4+ and CD8+ T cells in the tumor microenvironment. These results indicated that PLP could achieve immunotherapeutic effects and improve immunity in the breast tumor model.

Identification of a proline-kinked amphipathic α-helix downstream from the methyltransferase domain of a potexvirus replicase and its role in virus replication and perinuclear complex formation

Characterized positive-strand RNA viruses replicate in association with intracellular membranes. Regarding viruses in the genus Potexvirus , the mechanism by which their RNA-dependent RNA polymerase (replicase) associates with membranes is understudied. Here, by membrane flotation analyses of the replicase of plantago asiatica mosaic potexvirus (PlAMV), we identified a region in the methyltransferase (MET) domain as a membrane association determinant. An amphipathic α-helix was predicted downstream from the core region of the MET domain and hydrophobic amino acid residues were conserved in the helical sequences in replicases of other potexviruses. NMR analysis confirmed the amphipathic α-helical configuration and unveiled a kink caused by a highly conserved proline residue in the α-helix. Substitution of this proline residue and other hydrophobic and charged residues in the amphipathic α-helix abolished PlAMV replication. Ectopic expression of a GFP-fusion with the entire MET domain resulted in the formation of a large perinuclear complex, where virus replicase and RNA co-located during virus infection. Except for the proline substitution, the amino acid substitutions in the α-helix that abolished virus replication also prevented the formation of the large perinuclear complex by the respective GFP-MET fusion. Small intracellular punctate structures were observed for all GFP-MET fusions and in vitro high molecular weight complexes were formed by both replication-competent and -incompetent viral replicons, and thus were not sufficient for replication competence. We discuss the roles of the potexvirus-specific, proline-kinked amphipathic helical structure in virus replication and intracellular large complex and punctate structure formation. IMPORTANCE RNA viruses characteristically associate with intracellular membranes during replication. Although virus replicases are assumed to possess membrane-targeting properties, their membrane association domains generally remain unidentified or poorly characterized. Here, we identified a proline-kinked amphipathic α-helix structure downstream from the methyltransferase core domain of PlAMV replicase as a membrane association determinant. This helical sequence, which includes the proline residue, was conserved among potexviruses and related viruses in the order Tymovirales . Substitution of the proline residue but not the other residues necessary for replication allowed formation of a large perinuclear complex within cells resembling those formed by PlAMV replicase and RNA during virus replication. Our results demonstrate the role of the amphipathic α-helix in PlAMV replicase in a perinuclear complex formation and virus replication and that a perinuclear complex formation by the replicase alone will not necessarily indicate successful virus replication.