Endogenous Hormone Levels and Transcriptomic Analysis Reveal the Mechanisms of Bulbil Initiation in Pinellia ternata.

Pinellia ternata is a medicinal plant that has important pharmacological value, and the bulbils serve as the primary reproductive organ; however, the mechanisms underlying bulbil initiation remain unclear. Here, we characterized bulbil development via histological, transcriptomic, and targeted metabolomic analyses to unearth the intricate relationship between hormones, genes, and bulbil development. The results show that the bulbils initiate growth from the leaf axillary meristem (AM). In this stage, jasmonic acid (JA), abscisic acid (ABA), isopentenyl adenosine (IPA), and salicylic acid (SA) were highly enriched, while indole-3-acetic acid (IAA), zeatin, methyl jasmonate (MeJA), and 5-dexoxystrigol (5-DS) were notably decreased. Through OPLS-DA analysis, SA has emerged as the most crucial factor in initiating and positively regulating bulbil formation. Furthermore, a strong association between IPA and SA was observed during bulbil initiation. The transcriptional changes in IPT (Isopentenyltransferase), CRE1 (Cytokinin Response 1), A-ARR (Type-A Arabidopsis Response Regulator), B-ARR (Type-B Arabidopsis Response Regulator), AUX1 (Auxin Resistant 1), ARF (Auxin Response Factor), AUX/IAA (Auxin/Indole-3-acetic acid), GH3 (Gretchen Hagen 3), SAUR (Small Auxin Up RNA), GA2ox (Gibberellin 2-oxidase), GA20ox (Gibberellin 20-oxidase), AOS (Allene oxide synthase), AOC (Allene oxide cyclase), OPR (Oxophytodienoate Reductase), JMT (JA carboxy l Methyltransferase), COI1 (Coronatine Insensitive 1), JAZ (Jasmonate ZIM-domain), MYC2 (Myelocytomatosis 2), D27 (DWARF27), SMAX (Suppressor of MAX2), PAL (Phenylalanine Ammonia-Lyase), ICS (Isochorismate Synthase), NPR1 (Non-expressor of Pathogenesis-related Genes1), TGA (TGACG Sequence-specific Binding), PR-1 (Pathogenesis-related), MCSU (Molybdenium Cofactor Sulfurase), PP2C (Protein Phosphatase 2C), and SnRK (Sucrose Non-fermenting-related Protein Kinase 2) were highly correlated with hormone concentrations, indicating that bulbil initiation is coordinately controlled by multiple phytohormones. Notably, eight TFs (transcription factors) that regulate AM initiation have been identified as pivotal regulators of bulbil formation. Among these, WUS (WUSCHEL), CLV (CLAVATA), ATH1 (Arabidopsis Thaliana Homeobox Gene 1), and RAX (Regulator of Axillary meristems) have been observed to exhibit elevated expression levels. Conversely, LEAFY demonstrated contrasting expression patterns. The intricate expression profiles of these TFs are closely associated with the upregulated expression of KNOX(KNOTTED-like homeobox), suggesting a intricate regulatory network underlying the complex process of bulbil initiation. This study offers a profound understanding of the bulbil initiation process and could potentially aid in refining molecular breeding techniques specific to P. ternata.

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.

Cytokinin Type-B Response Regulators Promote Bulbil Initiation in Lilium lancifolium.

The bulbil is an important vegetative reproductive organ in triploid Lilium lancifolium whose development is promoted by cytokinins. Type-B response regulators (RRs) are critical regulators that mediate primary cytokinin responses and promote cytokinin-induced gene expression. However, the function of cytokinin type-B Arabidopsis RRs (ARRs) in regulating bulbil formation is unclear. In this study, we identified five type-B LlRRs, LlRR1, LlRR2, LlRR10, LlRR11 and LlRR12, in L. lancifolium for the first time. The five LlRRs encode proteins of 715, 675, 573, 582 and 647 amino acids. All of the regulators belong to the B-I subfamily, whose members typically contain a conserved CheY-homologous receiver (REC) domain and an Myb DNA-binding (MYB) domain at the N-terminus. As transcription factors, all five type-B LlRRs localize at the nucleus and are widely expressed in plant tissues, especially during axillary meristem (AM) formation. Functional analysis showed that type-B LlRRs are involved in bulbil formation in a functionally redundant manner and can activate LlRR9 expression. In summary, our study elucidates the process by which cytokinins regulate bulbil initiation in L. lancifolium through type-B LlRRs and lays a foundation for research on the molecular mechanism of bulbil formation in the lily.

Morphological and stage-specific transcriptome analyses reveal distinct regulatory programs underlying yam (Dioscorea alata L.) bulbil growth.

In yam (Dioscorea spp) species, bulbils at leaf axils are the most striking species-specific axillary structure and exhibit important ecological niches. Genetic regulation underlying bulbil growth remains largely unclear so far. Here, we characterize yam (Dioscorea alata L.) bulbil development using histological analysis, and perform full transcriptional profiling on key developmental stages together with phytohormone analyses. Using the stage-specific scoring algorithm, we have identified 3451 stage-specifically expressed genes that exhibit a tight link between major transcriptional changes and stages. Co-expressed gene clusters revealed an obvious over-representation of genes associated with cell division and expansion at the initiation stage of bulbils (T1). Transcriptional changes of hormone-related genes highly coincided with hormone levels, indicating that bulbil initiation and growth are coordinately controlled by multiple phytohormones. In particular, localized auxin is transiently required to trigger bulbil initiation, and be further depleted or exported from bulbils to promote growth by up-regulation of genes involved in auxinconjugation and efflux. The sharp increase in supply of sucrose and an enhanced trehalose-6-phophate pathway at T1 were observed, suggesting that sucrose probably functions as a key signal and promotes bulbil initiation. Analysis of the expression of transcription factors (TFs) predicated 149 TFs as stage-specifically expressed; several T1-specific TFs (from Aux/IAA, E2F, MYB, and bHLH families) have been shown to play key roles in triggering bulbil formation. Together, our work provides a crucial angle for in-depth understanding of the molecular programs underlying yam's unique bulbil development processes. Stage-specific gene sets can be queried to obtain key candidates regulating bulbil growth, serving as valuable resources for further functional research.

Mechanism of exogenous cytokinins inducing bulbil formation in Lilium lancifolium in vitro.

KEY MESSAGE: The cytokinin pathway promotes the initiation of bulbil formation, and iPA may an important type of cytokinin during bulbil formation in Lilium lancifolium. Bulbils are important vegetative reproductive organs in triploid Lilium lancifolium. We previously showed that cytokinins are involved in bulbil formation, but how cytokinins participate in bulbil formation is not clear. In this study, bulbil formation was divided into three stages on the basis of anatomical and histological observations: the bulbil initiation stage, bulbil primordium-formation stage and bulbil structure-formation stage. The results indicated that iPA was the most critical cytokinin during the bulbil initiation. qRT-PCR revealed that increased iPA content during bulbil initiation was mainly due to increased expression of cytokinin synthesis genes (IPT1/5) and cytokinin activation genes (LOG1/3/5/7) and significantly decreased expression of the cytokinin degradation gene CKX4. Exogenous 6-BA and lovastatin affected the cytokinin pathway and promoted or inhibited bulbil initiation by increasing or decreasing the content of endogenous iPA, respectively. In summary, we demonstrate that cytokinins positively regulate bulbil formation and provide preliminary insight into the regulatory mechanisms by which the cytokinin pathway promotes bulbil initiation.

Comparison of Structural and Functional Properties of Starches from the Rhizome and Bulbil of Chinese Yam.

Chinese yam is an important edible starch plant and widely cultivated in China. Its rhizome and bulbil are starch storage tissues below and above ground, respectively. In this paper, starches were isolated from the rhizome and bulbil of Chinese yam, and their structural and functional properties were compared. Both starches had an oval shape with an eccentric hilum and a CA-type crystalline structure. Their short-range ordered structure and lamellar structure had no significant difference. However, the rhizome starch had a significantly bigger granule size and lower amylose content than the bulbil starch. The swelling power and water solubility were significantly lower in the rhizome starch than in the bulbil starch. The onset and peak gelatinization temperatures were significantly higher in the rhizome starch than in the bulbil starch. The rhizome starch had a significantly higher breakdown viscosity and a lower setback viscosity than the bulbil starch. The thermal stability was lower in the rhizome starch than in the bulbil starch. The rhizome starch had a significantly lower resistance to hydrolysis and in vitro digestion than the bulbil starch. The above results provide important information for the utilization of rhizome and bulbil starches of Chinese yam.

Arabidopsis thaliana gonidialess A/Zuotin related factors (GlsA/ZRF) are essential for maintenance of meristem integrity.

Observation of a differential expression pattern, including strong expression in meristematic tissue of an Agave tequilana GlsA/ZRF ortholog suggested an important role for this gene during bulbil formation and developmental changes in this species. In order to better understand this role, the two GlsA/ZFR orthologs present in the genome of Arabidopsis thaliana were functionally characterized by analyzing expression patterns, double mutant phenotypes, promoter-GUS fusions and expression of hormone related or meristem marker genes. Patterns of expression for A. thaliana show that GlsA/ZFR genes are strongly expressed in SAMs and RAMs in mature plants and developing embryos and double mutants showed multiple changes in morphology related to both SAM and RAM tissues. Typical double mutants showed stunted growth of aerial and root tissue, formation of multiple ectopic meristems and effects on cotyledons, leaves and flowers. The KNOX genes STM and BP were overexpressed in double mutants whereas CLV3, WUSCHEL and AS1 were repressed and lack of AtGlsA expression was also associated with changes in localization of auxin and cytokinin. These results suggest that GlsA/ZFR is an essential component of the machinery that maintains the integrity of SAM and RAM tissue and underline the potential to identify new genes or gene functions based on observations in non-model plants.

Functionally different PIN proteins control auxin flux during bulbil development in Agave tequilana.

In Agave tequilana, reproductive failure or inadequate flower development stimulates the formation of vegetative bulbils at the bracteoles, ensuring survival in a hostile environment. Little is known about the signals that trigger this probably unique phenomenon in agave species. Here we report that auxin plays a central role in bulbil development and show that the localization of PIN1-related proteins is consistent with altered auxin transport during this process. Analysis of agave transcriptome data led to the identification of the A. tequilana orthologue of PIN1 (denoted AtqPIN1) and a second closely related gene from a distinct clade reported as 'Sister of PIN1' (denoted AtqSoPIN1). Quantitative real-time reverse transcription-PCR (RT-qPCR) analysis showed different patterns of expression for each gene during bulbil formation, and heterologous expression of the A. tequilana PIN1 and SoPIN1 genes in Arabidopsis thaliana confirmed functional differences between these genes. Although no free auxin was detected in induced pedicel samples, changes in the levels of auxin precursors were observed. Taken as a whole, the data support the model that AtqPIN1 and AtqSoPIN1 have co-ordinated but distinct functions in relation to auxin transport during the initial stages of bulbil formation.

Bulbil initiation: a comprehensive review on resources, development, and utilisation, with emphasis on molecular mechanisms, advanced technologies, and future prospects.

Bulbil is an important asexual reproductive structure of bulbil plants. It mainly grows in leaf axils, leaf forks, tubers and the upper and near ground ends of flower stems of plants. They play a significant role in the reproduction of numerous herbaceous plant species by serving as agents of plant propagation, energy reserves, and survival mechanisms in adverse environmental conditions. Despite extensive research on bulbil-plants regarding their resources, development mechanisms, and utilisation, a comprehensive review of bulbil is lacking, hindering progress in exploiting bulbil resources. This paper provides a systematic overview of bulbil research, including bulbil-plant resources, identification of development stages and maturity of bulbils, cellular and molecular mechanisms of bulbil development, factors influencing bulbil development, gene research related to bulbil development, multi-bulbil phenomenon and its significance, medicinal value of bulbils, breeding value of bulbils, and the application of plant tissue culture technology in bulbil production. The application value of the Temporary Immersion Bioreactor System (TIBS) and Terahertz (THz) in bulbil breeding is also discussed, offering a comprehensive blueprint for further bulbil resource development. Additionally, additive, seven areas that require attention are proposed: (1) Utilization of modern network technologies, such as plant recognition apps or websites, to collect and identify bulbous plant resources efficiently and extensively; (2) Further research on cell and tissue structures that influence bulb cell development; (3) Investigation of the network regulatory relationship between genes, proteins, metabolites, and epigenetics in bulbil development; (4) Exploration of the potential utilization value of multiple sprouts, including medicinal, ecological, and horticultural applications; (5) Innovation and optimization of the plant tissue culture system for bulbils; (6) Comprehensive application research of TIBS for large-scale expansion of bulbil production; (7) To find out the common share genetics between bulbils and flowers.

Bulbils of Aerial Yam Attenuate Ethanol-Induced Hepatotoxicity in HepG2 Cells through Inhibition of Oxidative Stress by Activation of the Nuclear Factor Erythroid-2-Related Factor 2 Signaling Pathway.

Bulbil of yam (BY) extract contains various active compounds possessing many pharmacological properties. However, little is known about the effect and underlying mechanism of BY extract on ethanol-induced liver damage. The present study explored the beneficial potential of BY extract on ethanol-induced hepatotoxicity. To evaluate its effectiveness, ethanol-induced HepG2 liver cells were pretreated with BY extract. BY extract effectively rescued cells from ethanol treatment through inhibition of apoptotic cell death as well as inhibiting expression of mitogen-activated protein kinase (MAPK) proteins as stress inducers. BY extract increased the expression of typical antioxidants. Furthermore, BY extract significantly inhibited mitochondrial dysfunction and endoplasmic reticulum (ER) stress, which are major ROS-inducing factors. Finally, as an underlying mechanism of the protective effects of BY extract on ethanol-induced liver damage, it activated Nrf2 protein through translocation from the cytosol to the nucleus, which in turn activated its target oxidative stress suppressor genes. Collectively, our findings demonstrate that BY extract has potential antioxidative effects in ethanol-induced liver cells and contributes to the establishment of a treatment strategy for alcohol-derived liver injuries.

mRNA-Seq and miRNA-Seq Analyses Provide Insights into the Mechanism of Pinellia ternata Bulbil Initiation Induced by Phytohormones.

Pinellia ternata (Thunb.) Breit (abbreviated as P. ternata) is a plant with an important medicinal value whose yield is restricted by many factors, such as low reproductive efficiency and continuous cropping obstacles. As an essential breeding material for P. ternata growth and production, the bulbils have significant advantages such as a high survival rate and short breeding cycles. However, the location effect, influencing factors, and molecular mechanism of bulbil occurrence and formation have not been fully explored. In this study, exogenously applied phytohormones were used to induce in vitro petiole of P. ternata to produce bulbil structure. Transcriptome sequencing of mRNA and miRNA were performed in the induced petiole (TCp) and the induced bulbil (TCb). Gene Ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed for the identification of key genes and pathways involved in bulbil development. A total of 58,019 differentially expressed genes (DEGs) were identified. The GO and KEGG analysis indicated that DEGs were mainly enriched in plant hormone signal transduction and the starch and sucrose metabolism pathway. The expression profiles of miR167a, miR171a, and miR156a during bulbil induction were verified by qRT-PCR, indicating that these three miRNAs and their target genes may be involved in the process of bulbil induction and play an important role. However, further molecular biological experiments are required to confirm the functions of the identified bulbil development-related miRNAs and targets.

Seed bulb size influences the effects of exogenous brassinolide on yield and quality of Pinellia ternata.

In recent years, natural Pinellia ternata populations of have gradually been exhausted, while the cultivated yield has been limited due to lack of research and uncertain climate condition. Therefore, it is necessary to explore methods of improving yield and quality in P. ternata using brassinolide (BR) treatments and choice of a suitable seed bulb size. This article reports the effects of BR and two seed bulb sizes (diameter: 0.5-1.0 cm and 1.0-1.5 cm) on active and nutrient components and antioxidant activity in P. ternata. The experiment included six levels of BR (0, 0.05, 0.10, 0.50, 1.00 and 2.00 mg l-1 ). The tuber yield of the two seed bulb sizes and bulbil yield of small seed bulbs increased 5.67%, 22.66% and 69.23% by day 105 after 0.50 mg l-1 BR treatment, compared with the control. On day 105, only 0.05 mg l-1 BR increased scores in principal components analysis (PCA) in tubers of small seed bulbs by 167.29%, and 0.05 and 0.50 mg l-1 BR increased PCA score in bulbils of large seed bulbs by 145.66% and 252.97%, respectively, compared with the control. Significant BR × seed bulb size interactions were found on yield and quality of P. ternata. The results indicate that BR effects on yield and quality of tubers and bulbils of P. ternata are not only related to BR concentration but also to seed bulb size.

Regulation Mechanism of Exogenous Brassinolide on Bulbil Formation and Development in Pinellia ternata.

The bulbil is the propagative organ of the P. ternata, which has a great effect on the yield of P. ternata. It is well known that plant hormones play important roles in bulbil formation and development. However, there is not clear about brassinolide (BR) regulation on bulbil formation and development. In this study, we revealed the effects of BR and BR biosynthesis inhibitors (propiconazole, Pcz) application on the histological observation, starch and sucrose metabolism, photosynthesis pathway, and hormone signaling pathway of P. ternata. The results showed that BR treatment reduced starch catabolism to maltodextrin and maltose in bulbil by decreasing BAM and ISA genes expression and increased cellulose catabolism to D-glucose in bulbil by enhancing edg and BGL genes expression. BR treatment enhanced the photosynthetic pigment content and potential maximum photosynthetic capacity and improved the photoprotection ability of P. ternata by increasing the dissipation of excess light energy to heat, thus reduced the photodamage in the PSII center. BR treatment increased the GA and BR content in bulbil of P. ternata, and decreased the ABA content in bulbil of P. ternata. Pcz treatment increased the level of GA, SL, ABA, and IAA in bulbil of P. ternata. BR regulated the signal transduction of BR, IAA, and ABA to regulate the formation and development of bulbil in P. ternata. These results provide molecular insight into BR regulation on bulbil formation and development.

Cloning and expression analysis of LoCCD8 during IAA-induced bulbils outgrowth in lily (Oriental Hybrid 'Sorbonne').

Aerial bulbils, which resemble tiny bulbs, develop from axillary buds and facilitate rapid propagation of Lilium. In most species of lily, bulbils are perpetually dormant and little is known about induction of these vegetative structures. Herein, we proposed that strigolactones (SLs) may regulate the induction of bulbils in Lilium. We tested this hypothesis by isolating and investigating the expression patterns of 2 copies of the carotenoid cleavage dioxygenase8 (CCD8) gene in lily-LoCCD8a and LoCCD8b-with regard to biosynthesis of SLs. Expression analyses revealed that LoCCD8a principally is expressed during vegetative growth, whereas LoCCD8b mainly is expressed during reproductive growth. The maximum quantity of LoCCD8a transcripts was observed in the basal plate in most developmental stages, which suggests that SLs may originate from underground parts, especially the basal plate, and move upward. The effects of treatments with indole-3-acetic acid or SL analog (GR24) on outgrowth of bulbils and expression of LoCCD8 genes suggested that SLs function downstream of auxin to inhibit the outgrowth of bulbil. The expression patterns of LoCCD8a and LoCCD8b at sprouting and bulblet weighting stages also implied that SLs may function in nutrient redistribution. Our findings are expected to promote the utilization of bulbils as vegetative propagules for commercial practice.

Histological and Transcriptomic Analysis during Bulbil Formation in Lilium lancifolium.

Aerial bulbils are an important propagative organ, playing an important role in population expansion. However, the detailed gene regulatory patterns and molecular mechanism underlying bulbil formation remain unclear. Triploid Lilium lancifolium, which develops many aerial bulbils on the leaf axils of middle-upper stem, is a useful species for investigating bulbil formation. To investigate the mechanism of bulbil formation in triploid L. lancifolium, we performed histological and transcriptomic analyses using samples of leaf axils located in the upper and lower stem of triploid L. lancifolium during bulbil formation. Histological results indicated that the bulbils of triploid L. lancifolium are derived from axillary meristems that initiate de novo from cells on the adaxial side of the petiole base. Transcriptomic analysis generated ~650 million high-quality reads and 11,871 differentially expressed genes (DEGs). Functional analysis showed that the DEGs were significantly enriched in starch and sucrose metabolism and plant hormone signal transduction. Starch synthesis and accumulation likely promoted the initiation of upper bulbils in triploid L. lancifolium. Hormone-associated pathways exhibited distinct patterns of change in each sample. Auxin likely promoted the initiation of bulbils and then inhibited further bulbil formation. High biosynthesis and low degradation of cytokinin might have led to bulbil formation in the upper leaf axil. The present study achieved a global transcriptomic analysis focused on gene expression changes and pathways' enrichment during upper bulbil formation in triploid L. lancifolium, laying a solid foundation for future molecular studies on bulbil formation.

Insurance against reproductive failure in a semelparous plant: bulbil formation in Agave macroacantha flowering stalks.

Bulbils are small aerial rosettes that occur on the flowering stalks of semelparous Agave plants and in related families, and that are capable of acting as clones of the parent plant. We hypothesized that bulbil formation was inversely related to fruiting success in the flowering stalk, and we explored this hypothesis in A. macroacantha, a species from South-Central Mexico. Forty randomly chosen plants were divided amongst three treatments: (a) elimination of all floral buds, (b) exclusion of pollinators, and (c) control. We also studied 22 plants in which the flowering stalk had been felled by goat grazing. Between September and November 1991 we kept a record of the numbers of bulbils and capsules produced in each flowering stalk. Significant (P<0.0001) differences between treatments were found in the proportion of plants hearing capsules and bearing bulbils. The control treatment had the highest proportion of plants producing capsules, treatment a had the highest proportion of individuals bearing bulbils, while treatment b showed an intermediate response. In the goat-grazed group, 45% of the plants failed to produce any propagative structure after the stalk was cut, and half of all plants produced bulbils on the remaining stump. A significant inverse relationship between the numbers of capsules and the numbers of bulbils per plant was found for the three randomly assigned treatments. Our results suggest that once the production of the flowering stalk has been triggered and the death of the rosette is irreversible, bulbils may act as an insurance mechanism that increases the probability of successful reproduction of the genet.