Neopolyploidy-induced changes in giant duckweed (Spirodela polyrhiza) alter herbivore preference and performance and plant population performance.

PREMISE: Polyploidy is a widespread mutational process in angiosperms that may alter population performance of not only plants but also their interacting species. Yet, knowledge of whether polyploidy affects plant-herbivore dynamics is scarce. Here, we tested whether aphid herbivores exhibit preference for diploid or neopolyploid plants, whether polyploidy impacts plant and herbivore performance, and whether these interactions depend on the plant genetic background. METHODS: Using independently synthesized neotetraploid strains paired with their diploid progenitors of greater duckweed (Spirodela polyrhiza), we evaluated the effect of neopolyploidy on duckweed's interaction with the water-lily aphid (Rhopalosiphum nymphaeae). Using paired-choice experiments, we evaluated feeding preference of the herbivore. We then evaluated the consequences of polyploidy on aphid and plant performance by measuring population growth over multiple generations. RESULTS: Aphids preferred neopolyploids when plants were provided at equal abundances but not at equal surface areas, suggesting the role of plant population surface area in driving this preference. Additionally, neopolyploidy increased aphid population performance, but this result was dependent on the plant's genetic lineage. Lastly, the impact of herbivory on neopolyploid vs. diploid duckweed varied greatly with genetic lineage, where neopolyploids appeared to be variably tolerant compared to diploids, sometimes mirroring the effect on herbivore performance. CONCLUSIONS: By experimentally testing the impacts of polyploidy on trophic species interactions, we showed that polyploidization can impact the preference and performance of herbivores on their plant hosts. These results have significant implications for the establishment and persistence of plants and herbivores in the face of plant polyploidy.

First Report of Leaf Rot Caused by Apiospora paraphaeosperma on Lily in Wuhan, China.

Lily (Lilium spp.) is a valuable ornamental bulb flower plant in Liliaceae, and its bulbs have high edible and medicinal value. Compared with bulb propagation of other lilies, seed propagation and short growth period are the most significant characteristics of Lilium×formolongi. In 2023, leaf rot disease (LRD) was observed on approximately 70% of the Lilium×formolongi seedlings sown in an experimental greenhouse in Wuhan, Hubei province, China. Irregular brown water-soaked spots were discovered in the early stages of infected seedlings. Then, spots spread throughout the leaves and caused the leaves to brown, soften, and wilted. A pathogen associated with symptoms was isolated by incubating sterilized leaves on potato dextrose agar plates at 25 ℃ for 2-3 days. Then, a pure single colony was isolated through a single hyphal tip isolation method. The fungal colony was white with abundant aerial mycelium and produced a yellow pigment diffusible into the agar. Microscopically, isolated mycelia were reticulate and pale yellow, while conidia were dark brown, smooth, and spherical, 7.31 to 6.98 × 4.03 to 3.87µm (average 5.44×5.41µm; n=30); oval in lateral view, and had a light stripe in the middle. To identify the species of the fungus at the molecular level, ITS and EF-1α genes were amplified and sequenced using primers ITS1/ITS4 (M Gardes et al. 1993) and 758F/986R (Carbone and Kohn 1999). The BLAST results in GenBank showed that the ITS(OR523578) and EF-1α(PP066842) sequences of LRD shared 99.82% and 99.24% identity with the distinct Apiospora paraphaeosperma strains (GenBank accession MT040110, ON806628.1, respectively). Combined with the morphology of the colony and conidium, the fungus was identified as Ap. paraphaeosperma. In the pathogenicity test, six healthy leaves were inoculated with mycelium disc and then kept in an incubator (22 ℃, 90% humidity, 16h light /8h darkness). The inoculated leaves showed necrosis and wilt symptoms similar to those observed in the greenhouse, while the control leaves were asymptomatic. A re-isolation, morphology identification and DNA sequencing of the fungus confirmed its infection with Ap. paraphaeosperma in Lilium spp. At present, rot caused by Ap. paraphaeosperma has only been reported in Thailand and South Korea, both of which are found on bamboo stems (Hyde et al. 2016; Sun Lul Kwon et al. 2022). As far as we know, this is the first report of leaf rot of lily caused by Ap. paraphaeosperma in China. This report can help identify this disease and further develop effective control measures.

First report of Fusarium commune Causing Bulb Rot on Lily (Lilium brownii var. Viridulum Baker) in Hunan Province of China.

Lily (Lilium brownii var. Viridulum Baker) is a well-known edible plant with large, white and sweet bulb scales that has important medicinal value (Zhou et al. 2021) and is grown mainly in the Hebei, Shanxi and Henan provinces of China. In May 2021, a case of bulb rot was discovered in a 3.33 hm2 plantation in Huaihua, Hunan Province, affecting 20% of the area (27°59'30″N, 110°32'20″E). The disease is most severe during the rainy season in May and June. In the early stage, irregular brown spots appeared on the lily scales, the necrosis was depressed and gradually enlarges, and in the later stage, the scales were scattered from the base of the disc and slough off. Ten samples were taken randomly from different plants in the plantation area to isolate the pathogens. After washing with sterile water, they were cut into small pieces and sterilised with 3% hydrogen peroxide for 30 s, 75% ethanol for 90 s, rinsed three times with sterile water and dried on sterile filter paper, then placed on a water agar plate and incubated in the dark in a constant temperature incubator at 28℃ for 3 to 5 days. After 2 days, the mycelium at the edge of the colony was transferred to a PDA plate and incubated for 3-5 days at 28°C in the dark to obtain pure fungal isolates. Eighteen purified fungal isolates were obtained, of which sixteen looked like Fusarium (88.9% isolation rate) and three representative isolates (BHBR2, BHBR3 and BHBR5) were selected for further study. The surface of this fungus was white with dense aerial mycelium. Some had an orange centre in the medium. Microconidia were oval in shape and appeared either straight or slightly curved. These microconidia were colourless, had 0-1 septa and measured 3.334 to 14.724 × 2.216 to 5.385 µm (n=100). Macroconidia were predominantly three-septate, crescent-shaped structures that were thin-walled and slightly curved. Cells at the apex and base were similarly curved. Macroconidia measured 17.956 to 32.150 × 2.788 to 4.492 µm (n=100). The mitochondrial small subunit (mtSSU) and translation elongation factor 1-α (TEF1) genes were amplified and sequenced using the NMS1/NMS2 and TEF-R/TEF-F primers to verify the identity of the pathogens (Stewart et al. 2006). The sequences were submitted to GenBank (BHBR2: mtSSU, PP273435; TEF, OR900976; BHBR3: mtSSU, PP277729; TEF, OR900977; BHBR5: mtSSU, PP277728; TEF, OR900978). A concatenated phylogenetic tree of the two genes was constructed and analysis showed that BHBR2, BHBR3 and BHBR5 were significantly clustered with Fusarium commune. Based on the results of morphological identification and phylogenetic tree analysis, the three isolates were identified as Fusarium commune. We carried out pathogenicity tests using two methods, one in which 6 × 6 mm fungal blocks were inoculated on lily (L. brownie var. viridulum Baker) scales and controls inoculated with sterile blocks, and the other in which strain BHBR2 was selected to carry out pathogenicity tests on bulbs of live plants soaked with 50 ml of a 1 × 106 conidial suspension and bulbs of control plants soaked with sterile water, all in three replicates. They were placed in a growth chamber at 28°C and 80% relative humidity, and the scales were moistened with moistened sterile filter paper. After 3 days of rearing treated scales, lesions appeared on lily scales inoculated with mycelial blocks and expanded with time, whereas no lesions appeared on lily scales inoculated with sterile blocks. One month later, whole plants soaked in the spore suspension wilted, while the control plants grew well. The pathogens re-isolated from the diseased tissues had the same morphological characteristics as representative isolates. This confirms Koch's hypothesis. Fusarium commune has been shown to be the most important pathogenic fungus causing root rot in Alfalfa (Medicago sativa) (Yang et al. 2022) and blueberry (Vaccinium uliginosum L.) (Li et al. 2023) in China. To our knowledge, this is the first report of Fusarium commune causing lily bulb rot in the world, which will lay the foundation for future control of lily bulb rot.

Comparative transcriptome analysis reveals the regulatory mechanisms of two tropical water lilies in response to cold stress.

BACKGROUND: Tropical water lily is an aquatic plant with high ornamental value, but it cannot overwinter naturally at high latitudes. The temperature drop has become a key factor restricting the development and promotion of the industry. RESULTS: The responses of Nymphaea lotus and Nymphaea rubra to cold stress were analyzed from the perspective of physiology and transcriptomics. Under the cold stress, Nymphaea rubra had obvious leaf edge curling and chlorosis. The degree of peroxidation of its membrane was higher than that of Nymphaea lotus, and the content of photosynthetic pigments also decreased more than that of Nymphaea lotus. The soluble sugar content, SOD enzyme activity and CAT enzyme activity of Nymphaea lotus were higher than those of Nymphaea rubra. This indicated that there were significant differences in the cold sensitivity of the two varieties. GO enrichment and KEGG pathway analysis showed that many stress response genes and pathways were affected and enriched to varying degrees under the cold stress, especially plant hormone signal transduction, metabolic pathways and some transcription factor genes were from ZAT gene family or WKRY gene family. The key transcription factor ZAT12 protein in the cold stress response process has a C2H2 conserved domain, and the protein is localized in the nucleus. Under the cold stress, overexpression of the NlZAT12 gene in Arabidopsis thaliana increased the expression of some cold-responsive protein genes. The content of reactive oxygen species and MDA in transgenic Arabidopsis thaliana was lower, and the content of soluble sugar was higher, indicating that overexpression of NlZAT12 can improve the cold tolerance of Arabidopsis thaliana. CONCLUSION: We demonstrate that ethylene signalling and reactive oxygen species signalling play critical roles in the response of the two cultivars to cold stress. The key gene NlZAT12 for improving cold tolerance was identified. Our study provides a theoretical basis for revealing the molecular mechanism of tropical water lily in response to cold stress.

Low LdMYB12 expression contributes to petal spot deficiency in Lilium davidii var. unicolor.

Petal spots are widespread in plants, they are important for attracting pollinators and as economic traits in crop breeding. However, the genetic and developmental control of petal spots has seldom been investigated. To further clarify the development of petal spots formation, we performed comparative transcriptome analysis of Lilium davidii var. unicolor and Lilium davidii petals at the full-bloom stage. In comparison with the parental species L. davidii, petals of the lily variety L. davidii var. unicolor do not have the distinct anthocyanin spots. We show that among 7846 differentially expressed genes detected, LdMYB12 was identified as a candidate gene contributing to spot formation in lily petals. The expression level of LdMYB12 in the petals of L. davidii was higher than that in L. davidii var. unicolor petals. Moreover, overexpression of LdMYB12 led to the appearance of spots on the petals of L. davidii var. unicolor, accompanied by increased expression of anthocyanin synthesis-related genes. Taken together, these results indicate that abnormal expression of LdMYB12 contributes to petal spot deficiency in L. davidii var. unicolor.

First report of dasheen mosaic virus infecting calla lilies in South Korea.

In April 2022, leaves showing virus-like symptoms including mosaic, feathery chlorotic mottle and distortions were observed on calla lilies (Zantedeschia sp.) growing in a greenhouse in Jeolla province, South Korea. Leaf samples from nine symptomatic plants from the same greenhouse were collected and tested for Zantedeschia mosaic virus (ZaMV), Zantedeschia mild mosaic virus (ZaMMV) and Dasheen mosaic virus (DaMV) by reverse transcription-polymerase chain reaction (RT-PCR) with specific primers, ZaMV-F/R (Wei et al. 2008), ZaMMV-F/R (5'-GACGATCAGCAACAGCAGCAACAGCAGAAG-3'/5'-CTGCAAGGCTGAGATCCCGAGTAGCGAGTG-3') and DsMV-CPF/CPR, respectively. In previous surveys, ZaMV and ZaMMV were detected in calla lily fields in South Korea. Of 9 symptomatic samples, 8 were positive for ZaMV and ZaMMV but no PCR product was obtained from the ninth sample, which showed a yellow feather-like pattern. To identify the causal virus, total RNA from a leaf sample of the symptomatic calla lily was extracted using an RNeasy Plant Mini Kit (Qiagen, Germany) and analyzed by high-throughput sequencing. Ribosomal RNA was removed and a cDNA library was prepared using an Illumina TruSeq Stranded Total RNA LT Sample Prep Kit (Plants) and sequenced on an Illumina NovaSeq 6000 system (Macrogen, Korea), yielding 150 nt paired end reads. De novo assembly of the 88,171,036 reads was performed using Trinity software (r20140717) while the 113,140 initially assembled contigs were screened against the NCBI viral genome database using BLASTN. One contig of 10,007 bp (GenBank LC723667) shared 79.89-87.08% nucleotide (nt) identities to the available genomes of other DsMV isolates including Colocasia esculenta isolates Et5 (MG602227, 87.08%; Ethiopia) and CTCRI-II-14 (KT026108, 85.32%; India), and a calla lily isolate (AJ298033, 84.95%; China). No contigs representing other plant viruses were identified. To confirm the presence of DsMV, and because the virus was not detected using DsMV-CPF/CPR, RT-PCR was performed using new virus-specific primers DsMV-F/R (5'-GATGTCAACGCTGGCACCAGT-3'/5'-CAACCTAGTAGTAACGTTGGAGA-3'), designed based on the contig sequence. PCR products of the expected 600 bp were obtained from the symptomatic plant, cloned into the pGEM-T Easy Vector (Promega, USA), and two independent clones were bidirectionally sequenced (BIONEER, Korea), and shown to be identical. The sequence was deposited in GenBank as acc. no. LC723766, and shared 100% nt identity to the full-length contig LC723667, and 91.83% identity to the Chinese calla lily DsMV isolate (AJ298033). DsMV, a member of the genus Potyvitus in the family Potyviridae, is one of the major viruses infecting taro in South Korea, showing mosaic and chlorotic feathering symptoms (Kim et al. 2004); however, there is no record in the literature of the identification of this virus in South Korea in ornamental species including calla lily. To survey the sanitary status of other calla lilies, 95 samples with or without symptoms were collected from other regions and subjected to RT-PCR detection for DsMV. Ten of these samples were positive with primers DsMV-F/R, including seven mixed infections (DsMV+ZaMV or DsMV+ZaMV+ZaMMV). To our knowledge, this is the first report of DsMV infecting calla lilies in South Korea. The virus is easily spread by vegetative propagation (Babu et al. 2011) and by aphids (Reyes et al. 2006). This study will help the management of viral diseases on calla lilies in South Korea.

First report of Lily virus X infecting Paris polyphylla var. yunnanensis in Yunnnan, China.

Lily virus X (LVX) is a positive-sense ssRNA virus belonging to the genus Potexvirus in the family Alphaflexiviridae. LVX is known to infect plants of the genera Lilium and Tricyrtis in the family Liliacea. LVX was first reported in an asymptomatic lily (Lilium formosanum) from England (Stone, 1980), but has been shown to infect plants in the Netherlands (Chen et al. 2005), the United States (Jordan et al. 2008) and Japan (Nijo et al. 2018). To date, the complete genomes of two LVX isolates from the Netherlands and Japan have been reported. Paris polyphylla var. yunnanensis, known as Dianchonglou in China, is a perennial plant of the family Melanthiaceae (formerly belonging to the family Trillium). In China, its rhizome is commonly used as an antispasmodic agent for stroke and cancer treatment (Chang et al. 2017). From 2019 to 2022, leaf mottle and shrinkage which are typical symptoms of viral infections were observed on the leaves of P. polyphylla var. yunnanensis plants in Dianchonglou fields in Qujing, Yunnan. Disease incidence ranged from 19% to 45% across 5 fields (90 plants per field) in Qujing. To identify the possible viral pathogen(s) associated with the disease, the mirVanaTM miRNA isolation Kit was used to extract total RNA was from a mixed sample pool of 5 symptomatic leaf samples collected from the 5 fields. RNA sequencing library was constructed using TruSeqTM RNA sample preparation kit. Sequencing on the Illumina HiSeqTM 2500 platform (Illumina, USA) with 125-bp paired-end reads yielded 23,077,786 raw reads. 22,534,100 clean reads were obtained by removing reads of low quality and poly-N using Trimmomatic software (Bolger et al. 2014). By utilizing the paired-end splicing method in Trinity software (Grabherr et al. 2011) the the raw reads were De novo assembled into 184,596 contigs, of which 303 were related to viruses, including Paris mosaic necrosis virus (PMNV), Pear alphapartitivirus (PAPV), Dahlia mosaic virus (DMV), and Lily virus X (LVX). BLASTn analysis revealed that 12 contigs (lengths ranging from 344 nt to 5,981 nt, query cover 6% to 99%) were most similar (57.32% to 91.67% nt identities) to the genome sequences of LVX, suggesting a possible infection of LVX in the plants. To confirm the result, a full-length genomic sequence of LVX was obtained by reverse transcription polymerase chain reaction (RT-PCR) using specific primers designed based on the sequence of the assembled contigs. The PCR products were cloned into pGEM-T vector (Promega Corporation, USA) and sequenced using the Sanger method (Sangon Biotech, Shanghai, China). The obtained full-length genomic sequence of the LVX isolate (LVX-PP, accession number OM100017) was 5,981 nt in length. BLASTp analysis demonstrated that the putative Rep and CP of LVX-PP shared 76.27% to 81.05% and 80.81% to 81.82% aa sequence similarities with that of other LVX isolates, respectively. Maximum-likelihood phylogenetic trees inferred from the Rep and CP aa sequences showed that LVX-PP clustered closely with LVX isolates. The leaf samples were further analyzed using a lily virus X (LVX) ELISA kit (DEIAPV181, Creative Diagnostics, U.S.A.). Healthy P. polyphylla var. yunnanensis leaves were taken as a negative control and buffer solution as a blank control. The results showed a positive reaction for all five symptomatic plants (OD = 1.259 ± 0.007) relative to the negative (OD = 0.099) and blank (OD = 0.073) controls. These results indicate that LVX can infect P. polyphylla var. yunnanensis. To our knowledge, this is the first report that LVX has been detected in P. polyphylla var. yunnannensis. This study will serve as an important reference for the study of the host range of LVX. Further studies will be required to determine how LVX spreads between P. polyphylla var. yunnannensis and other host plants.

Insights into Adaptive Regulation of the Leaf-Petiole System: Strategies for Survival of Water Lily Plants under Salt Stress.

The water lily (Nymphaea tetragona) is an ancient angiosperm that belongs to the Nymphaeaceae family. As a rooted floating-leaf plant, water lilies are generally cultivated in fresh water, therefore, little is known about their survival strategies under salt stress. Long-term salt stress causes morphological changes, such as the rapid regeneration of floating leaves and a significant decrease in leaf number and surface area. We demonstrate that salt stress induces toxicity soon after treatment, but plants can adapt by regenerating floating leaves that are photosynthetically active. Transcriptome profiling revealed that ion binding was one of the most-enriched GO terms in leaf-petiole systems under salt stress. Sodium-transporter-related genes were downregulated, whereas K+ transporter genes were both up- and downregulated. These results suggest that restricting intracellular Na+ importing while maintaining balanced K+ homeostasis is an adaptive strategy for tolerating long-term salt stress. ICP-MS analysis identified the petioles and leaves as Na-hyperaccumulators, with a maximum content of over 80 g kg-1 DW under salt stress. Mapping of the Na-hyperaccumulation trait onto the phylogenetic relationships revealed that water lily plants might have a long evolutionary history from ancient marine plants, or may have undergone historical ecological events from salt to fresh water. Ammonium transporter genes involved in nitrogen metabolism were downregulated, whereas NO3--related transporters were upregulated in both the leaves and petioles, suggesting a selective bias toward NO3- uptake under salt stress. The morphological changes we observed may be due to the reduced expression of genes related to auxin signal transduction. In conclusion, the floating leaves and submerged petioles of the water lily use a series of adaptive strategies to survive salt stress. These include the absorption and transport of ions and nutrients from the surrounding environments, and the ability to hyperaccumulate Na+. These adaptations may serve as the physiological basis for salt tolerance in water lily plants.

Chemical Composition, Market Survey, and Safety Assessment of Blue Lotus (Nymphaea caerulea Savigny) Extracts.

Blue lotus, also known as Nymphaea caerulea (Nymphaeaceae), is a water lily found globally in lakes and rivers. With its long history of use in Egyptian culture, blue lotus has been associated with spiritual rituals and health benefits. Nowadays, blue lotus is still consumed as a tea or tincture to induce relaxation and heightened spiritual awareness. In this study, six authentic N. caerulea extracts from trusted sources and eleven commercial products were analyzed using gas chromatography-mass spectrometry (GC-MS). Authentic blue lotus extracts were produced in industrial settings. Overall, the extracts were a mixture of aliphatic hydrocarbons, aromatic alcohols, fatty acids, phenyl derivatives, diterpenoids, phytosterols, and stigmastanes. Apomorphine and nuciferine, which are responsible for psychoactive effects of the blue lotus flower, were virtually absent from the authentic blue lotus extract. Although blue lotus has a long history of use, the safety data on the plant and its extracts is limited; however, together with the analytical data, the available information does not indicate major safety concerns for the topical application of authentic blue lotus flower concrete or absolute when diluted as a fragrance ingredient.

Phosphodiesterase 5 and Arginase Inhibitory Activities of the Extracts from Some Members of Nelumbonaceae and Nymphaeaceae Families.

The objectives of this study were (1) to investigate the effect of extracts from some plants in the families Nelumbonaceae and Nymphaeaceae on phosphodiesterase 5 (PDE5) and arginase, which have been used in erectile dysfunction treatment, and (2) to isolate and identify the compounds responsible for such activities. The characterization and quantitative analysis of flavonoid constituents in the active extracts were performed by HPLC. Thirty-seven ethanolic extracts from different parts of plants in the genus Nymphaea and Victoria of Nymphaeaceae and genus Nelumbo of Nelumbonaceae were screened for PDE5 and arginase inhibitory activities. The ethanolic extracts of the receptacles and pollens of Nelumbo nucifera Gaertn., petals of Nymphaea cyanea Roxb. ex G.Don, Nymphaea stellata Willd., and Victoria amazonica (Poepp.) Sowerby and the petals and receptacles of Nymphaea pubescens Willd. showed IC50 values on PDE5 of less than 25 µg/mL while none of the extracts showed effects on arginase. The most active extract, N. pubescens petal extract, was fractionated to isolate and identify the PDE5 inhibitors. The results showed that six flavonoid constituents including quercetin 3'-O-ß-xylopyranoside (1), quercetin 3-methyl ether 3'-O-ß-xylopyranoside (2), quercetin (3), 3-O-methylquercetin (4), kaempferol (5) and 3-O-methylkaempferol (6) inhibited PDE5 with IC50 values at the micromolar level.

Full-length transcriptome-referenced analysis reveals crucial roles of hormone and wounding during induction of aerial bulbils in lily.

Aerial bulbils are important vegetative reproductive organs in Lilium. They are often perpetually dormant in most Lilium species, and little is known about the induction of these vegetative structures. The world-famous Oriental hybrid lily cultivar 'Sorbonne', which blooms naturally devoid of aerial bulbils, is known for its lovely appearance and sweet fragrance. We found that decapitation stimulated the outgrowth of aerial bulbils at lower stems (LSs) and then application of low and high concentrations of IAA promoted aerial bulbils emergence around the wound at upper stems (USs) of 'Sorbonne'. However, the genetic basis of aerial bulbil induction is still unclear. Herein, 'Sorbonne' transcriptome has been sequenced for the first time using the combination of third-generation long-read and next-generation short-read technology. A total of 46,557 high-quality non-redundant full-length transcripts were generated. Transcriptomic profiling was performed on seven tissues and stems with treatments of decapitation and application of low and high concentrations of IAA, respectively. Functional annotation of 1918 DEGs within stem samples of different treatments showed that hormone signaling, sugar metabolism and wound-induced genes were crucial to bulbils outgrowth. The expression pattern of auxin-, shoot branching hormone-, plant defense hormone- and wound-inducing-related genes indicated their crucial roles in bulbil induction. Then we established five hormone- and wounding-regulated co-expression modules and identified some candidate transcriptional factors, such as MYB, bZIP, and bHLH, that may function in inducing bulbils. High connectivity was observed among hormone signaling genes, wound-induced genes, and some transcriptional factors, suggesting wound- and hormone-invoked signals exhibit extensive cross-talk and regulate bulbil initiation-associated genes via multilayered regulatory cascades. We propose that the induction of aerial bulbils at LSs after decapitation can be explained as the release of apical dominance. In contrast, the induction of aerial bulbils at the cut surface of USs after IAA application occurs via a process similar to callus formation. This study provides abundant candidate genes that will deepen our understanding of the regulation of bulbil outgrowth, paving the way for further molecular breeding of lily.

Complete genome organization and characterization of Hippeastrum latent virus.

The complete genome sequences of two carlaviruses were determined by high-throughput sequencing of RNA extracted from ringspot and mosaic, disease symptoms on leaves of spider lily plants (Crinum asiaticum, family Amaryllidaceae) growing as landscape plants in Hawaii. One, named Nerine latent virus (NeLV)-Hawaii with a genome of 8281 nucleotide exhibited the highest nucleotide identity and amino acid similarity of 95.5% and 96.0%, respectively, to the genome sequence of an isolate of NeLV from Narcissus sp. in Australia (JQ395044). The second, named Hippeastrum latent virus (HiLV)-Hawaii with a genome of 8497 nucleotides exhibited the highest nucleotide identity and amino acid similarity, 84.3% and 88.7%, respectively, to the sequence of a previously uncharacterized HiLV isolate from a potted flowering plant, Amaryllis (Hippeastrum hybridum Hort) in Taiwan (DQ098905). The amino acid sequence similarities of replicase (Rep) and coat protein (CP) between HiLV-Hawaii and NeLV-Hawaii were 44.8% and 38.4%, respectively. Results of viral protein Rep and CP amino acid sequence comparisons from various carlaviruses provide evidence that HiLV and NeLV, previously classified as synonymous viruses are in fact unique viruses. This is the first report for the complete sequence, organization, and phylogenetic characterization of HiLV and the first detection of HiLV both in C. asiaticum and in the USA.

Arthrobacter wenxiniae sp. nov., a novel plant growth-promoting rhizobacteria species harbouring a carotenoids biosynthetic gene cluster.

A bacterial strain, designated AETb3-4T was isolated from the rhizosphere of lily. Comparison of 16S rRNA gene sequences showed that the sequence from strain AETb3-4T exhibits high sequence similarity with those of Arthrobacter silviterrae KIS14-16T (97.9%), Arthrobacter livingstonensis LI2T (97.2%) and Arthrobacter stackebrandtii CCM 2783T (97.0%). Whole genome average nucleotide identity (ANI) and the digital DNA-DNA hybridization (dDDH) values between strain AETb3-4T and the reference strains A. silviterrae DSM 27180T, A. livingstonensis L12T and A. stackebrandtii DSM 16005T were below 83.6% and 27.7%, respectively, values which are considerably below the proposed thresholds for the species delineation, consistent with the proposal that strain AETb3-4T represents a novel species. The genome size of strain AETb3-4T is 4.33 Mb and the genomic DNA G + C content is 67.3%. The main polar lipids were identified as phosphatidylglycerol, diphosphatidylglycero, phosphatidylinositol and an unidentified glycolipid. The major fatty acids (> 10%) were identified as anteiso-C15: 0 and anteiso-C17: 0. The predominant menaquinone was found to be menaquinone 9 (MK-9) (H2) (82.2%). Phenotypic tests allowed the strain to be differentiated from its close phylogenetic neighbors. Based on the results obtained, it is proposed that the strain AETb3-4T (= CFCC 16390T = LMG 31708T) represents a novel species in the genus Arthrobacter, for which the names Arthrobacter wenxiniae sp. nov. is proposed. In addition, the novel strain AETb3-4T has multiple plant growth-promoting characters including ACC-deaminase activity and production of IAA. Furthermore, the genome contains secondary metabolite biosynthesis gene clusters, including a carotenoid biosynthetic gene cluster, suggesting potential capacities for secondary metabolite synthesis. These data suggest that strain AETb3-4T may have potential applications both in medicine and sustainable agriculture.

Comparative metabolome analysis unravels a close association between dormancy release and metabolic alteration induced by low temperature in lily bulbs.

KEY MESSAGE: The correlation between dormancy release and metabolic metabolic changes in lily bulbs during low temperature storage was investigated. Low temperature is a major environmental factor required for dormancy release in lily bulbs. Although great advances in plant metabolomics have been achieved, knowledge about the molecular basis of lily bulb metabolomes at different developmental stages in response to low temperature is still limited. In this work, the dormancy release, vegetative growth, flowering, metabolic profile and gene expression in the less dormant cultivar Lilium longiforum × Oriental hybrid 'Triumphator' (T) and the more dormant cultivar Lilium Asiatic hybrid 'Honesty' (H) were compared. Exposure to low temperature (LT) successfully promoted stem elongation, floral transition and flowering of both T and H bulbs. However, exposure to room temperature (RT) restricted stalk elongation of both T and H bulbs, and prohibited floral transition and flowering of H bulbs. Correspondingly, higher antioxidant enzyme activity and total primary metabolite contents were observed in the apical bud of T bulbs. Gene expression analysis revealed that expressions of LiFT, LiFLK, LiSOC1 and LiCBF were decreased, whereas the expression of LiSVP and LiFLC were increased, in the apical bud of H bulbs under RT storage condition. Our findings reveal that the growth and dormancy breaking of lily bulbs are closely associated with the metabolic changes in the apical buds during postharvest storage.

Isolation and functional diversity of Bowman-Birk type serine proteinase inhibitors from Hyacinthus orientalis.

Bowman-Birk inhibitors (BBIs) are plant-derived serine proteinase inhibitors. Endogenously, they function as defense molecules against pathogens and insects, but they also have been explored for applications in cancer treatment and inflammatory disorders. Here, we isolated 15 novel BBIs from the bulb of Hyacinthus orientalis (termed HOSPIs). These isoinhibitors consisted of two or three chains, respectively, that are linked by disulfides bonds based on proposed cleavage sites in the canonical BBI reactive site loop. They strongly inhibited trypsin (Ki = 0.22-167 nM) and α-chymotrypsin (Ki = 19-1200 nM). Notably, HOSPI-B4 contains a six-residue reactive loop, which appears to be the smallest such motif discovered in BBIs to date. HOSPI-A6 and -A7 contain an unusual reactive site, i.e. Leu-Met at the P1-P1' position and have strong inhibitory activity against trypsin, α-chymotrypsin, and elastase. Analysis of the cDNA encoding HOSPIs revealed that the precursors have HOSPI-like domains repeated at least twice with a defined linker sequence connecting individual domains. Lastly, mutational analysis of HOSPIs suggested that the linker sequence does not affect the inhibitory activity, and a Thr residue at the P2 site and a Pro at the P3' site are crucial for elastase inhibition. Using mammalian proteases as representative model system, we gain novel insight into the sequence diversity and proteolytic activity of plant BBI. These results may aid the rational design of BBI peptides with potent and distinct inhibitory activity against human, pathogen, or insect serine proteinases.

Starch Degradation and Sucrose Accumulation of Lily Bulbs after Cold Storage.

Functional lilies are a group of edible lily cultivars with great potential for landscape application. Low-temperature storage can significantly improve their taste, but the knowledge of this process is largely unknown. In this study, we used the functional lilies 'Fly Shaohua' and 'Fly Tiancheng' as materials. Through physiological observation and transcriptome analysis during the bulbs' cold storage, it was found that the starch degradation and sucrose accumulation in bulbs contributed to taste improvement. After 60 d of cold storage, the sucrose accumulation was highest and the starch content was lower in the bulbs, suggesting this time-point was optimal for consumption. Accompanying the fluctuation of sucrose content during cold storage, the enzyme activities of sucrose phosphate synthase and sucrose synthase for sucrose synthesis were increased. Transcriptome analysis showed that many differentially expressed genes (DEGs) were involved in the starch and sucrose metabolism pathway, which might promote the conversion of starch to sucrose in bulbs. In addition, the DEGs involved in dormancy and stress response were also determined during cold storage, which might explain the decreased sucrose accumulation with extended storage time over 60 d due to the energy consumption for dormancy release. Taken together, our results indicated sucrose accumulation was a main factor in the taste improvement of lily bulbs after cold storage, which is attributable to the different gene expression of starch and sucrose metabolism pathways in this process.

LoSWEET14, a Sugar Transporter in Lily, Is Regulated by Transcription Factor LoABF2 to Participate in the ABA Signaling Pathway and Enhance Tolerance to Multiple Abiotic Stresses in Tobacco.

Sugar transport and distribution plays an important role in lily bulb development and resistance to abiotic stresses. In this study, a member of the Sugar Will Eventually be Exported Transporters (SWEET) gene family, LoSWEET14, from Oriental hybrid lily 'Sorbonne' was identified. LoSWEET14 encodes a protein of 278 amino acids and is capable of transporting sucrose and some types of hexoses. The transcript level of the LoSWEET14 gene was significantly increased under various stress conditions including drought, cold, salt stresses, and abscisic acid (ABA) treatment. Overexpression of LoSWEET14 in tobacco (Nicotiana tabacum) showed that the transgenic lines had larger leaves, accumulated more soluble sugars, and were more resistant to drought, cold, and salt stresses, while becoming more sensitive to ABA compared with wild-type lines. Promoter analysis revealed that multiple stress-related cis-acting elements were found in the promoter of LoSWEET14. According to the distribution of cis-acting elements, different lengths of 5'-deletion fragments were constructed and the LoSWEET14-pro3(-540 bp) was found to be able to drive GUS gene expression in response to abiotic stresses and ABA treatment. Furthermore, a yeast one hybrid (Y1H) assay proved that the AREB/ABF (ABRE-binding protein/ABRE-binding factor) from lilies (LoABF2) could bind to the promoter of LoSWEET14. These findings indicated that LoSWEET14 is induced by LoABF2 to participate in the ABA signaling pathway to promote soluble sugar accumulation in response to multiple abiotic stresses.

Expression Patterns of Sugar Transporter Genes in the Allocation of Assimilates and Abiotic Stress in Lily.

During the growth cycle of lilies, assimilates undergo a process of accumulation, consumption and reaccumulation in bulbs and are transported and allocated between aboveground and underground organs and tissues. The sink-source relationship changes with the allocation of assimilates, affecting the vegetative growth and morphological establishment of lilies. In this study, the carbohydrate contents in different tissues of five critical stages during lily development were measured to observe the assimilates allocation. The results showed bulbs acted as the main source to provide energy before the budding stage (S3); after the flowering stage (S4), bulbs began to accumulate assimilates as a sink organ again. During the period when the plant height was 30cm with leaf-spread (S2), leaves mainly accumulated assimilates from bulbs through the symplastic pathway, while when leaves were fully expanded, it transformed to export carbohydrates. At the S4 stage, flowers became a new active sink with assimilates influx. To further understand the allocation of assimilates, 16 genes related to sugar transport and metabolism (ST genes) were identified and categorized into different subfamilies based on the phylogenetic analysis, and their protein physicochemical properties were also predicted. Tissue-specific analysis showed that most of the genes were highly expressed in stems and petals, and it was mainly the MST (monosaccharide transporter) genes that were obviously expressed in petals during the S4 stage, suggesting that they may be associated with the accumulation of carbohydrates in flowers and thus affect flower development process. LoSWEET14 (the Sugar will eventually be exported transporters) was significantly correlated with starch in scales and with soluble sugar in leaves. Sugar transporters LoHXT6 and LoSUT1 were significantly correlated with soluble sugar and sucrose in leaves, suggesting that these genes may play key roles in the accumulation and transportation of assimilates in lilies. In addition, we analyzed the expression patterns of ST genes under different abiotic stresses, and the results showed that all genes were significantly upregulated. This study lays a solid foundation for further research on molecular mechanism of sink-source change and response to abiotic stresses in lilies.

High promoter sequence variation in subgroup 6 members of R2R3-MYB genes is involved in different floral anthocyanin color patterns in Lilium spp.

The spatially and temporally distinct expression of R2R3-MYB positive regulators is among the major mechanisms that create various anthocyanin color patterns in many flowers. However, we do not know how these positive regulators have gained different expression profiles. In the Asiatic hybrid lily 'Lollypop' (derived from the crosses of species belonging to Sinomartagon/Daurolirion section), MYB12 and MYB19S regulate the pigmentation at whole tepals and raised tepal spots, respectively. In the Oriental hybrid lily 'Sorbonne' (derived from the crosses of species belonging to the Archelirion section), MYB12 regulates both whole tepal and raised spot pigmentation. The genes have similar amino acid sequences with similar protein functions but exhibit different expression profiles in lily flowers. As promoters are among the most significant factors affecting gene expression profiles, their promoter sequences were determined in this study. The three genes had very different promoter sequences, and putative cis-regulatory elements were not conserved in numbers or order. To further confirm the promoter functions, tobacco plants were transformed with native promoter-driven MYB12 or MYB19S genes of 'Lollypop.' Expression levels of MYB12 were higher in corolla tubes than in lobes, while those of MYB19S were higher in corolla lobes than in tubes. Thus, the diverse promoter functions were likely to be the leading causes of their different expression profiles and generation of unique color patterns. Finally, the history of R2R3-MYB gene establishment during lily evolution was estimated using sequence data.

Transcriptome profiling reveals key genes in regulation of the tepal trichome development in Lilium pumilum D.C.

KEY MESSAGE: A number of potential genes and pathways involved in tepal trichome development were identified in a natural lily mutant by transcriptome analysis and were confirmed with trichome and trichomeless species. Trichome is a specialized structure found on the surface of the plant with an important function in survival against abiotic and biotic stress. It is also an important economic trait in crop breeding. Extensive research has investigated the foliar trichome in model plants (Arabidopsis and tomato). However, the developmental mechanism of tepal trichome remains elusive. Lilium pumilum is an edible ornamental bulb and a good breeding parent possessing cold and salt-alkali resistance. Here, we found a natural mutant of Lilium pumilum grown on a highland whose tepals are covered by trichomes. Our data indicate that trichomes of the mutant are multicellular and branchless. Notably, stomata are also developed on the tepal of the mutant as well, suggesting there may be a correlation between trichome and stomata regulation. Furthermore, we isolated 27 differentially expressed genes (DEGs) by comparing the transcriptome profiling between the natural mutant and the wild type. These 27 genes belong to 4 groups: epidermal cell cycle and division, trichome morphogenesis, stress response, and transcription factors. Quantitative real-time PCR in Lilium pumilum (natural mutant and the wild type) and other lily species (Lilium leichtlinii var. maximowiczii/trichome; Lilium davidii var. willmottiae/, trichomeless) confirmed the validation of RNA-seq data and identified several trichome-related genes.