The A subunit of vacuolar H+-ATPase gene (CmVHA-A) plays opposite roles in plant growth and drought tolerance of chrysanthemum under different growing conditions.

As the most prominent proton pumps in plants, vacuolar H+-ATPases (VHAs) comprise multiple subunits that are important for physiological processes and stress tolerance in plants. However, few studies on the roles of subunit genes of VHAs in chrysanthemum have been reported to date. In this study, the gene of A subunit of V-ATPase in chrysanthemum (CmVHA-A) was cloned and identified. CmVHA-A was conserved with VHA-A proteins from other plants. Expression analysis showed that CmVHA-A was highly expressed in most tissues of chrysanthemum except for the flower bud, and was readily induced by polyethylene glycol (PEG) treatment. Functional analysis demonstrated that CmVHA-A exerted a negative influence on the growth and development of shoot and root of chrysanthemum under normal conditions. RNA-sequencing (RNA-seq) analysis revealed the possible explanations for phenotypic differences between transgenic and wild-type (WT) plants. Under drought conditions, CmVHA-A positively affected the drought tolerance of chrysanthemum by enhancing antioxidase activity and alleviating photosynthetic disruption. Overall, CmVHA-A plays opposite roles in plant growth and drought tolerance of chrysanthemums under different growing conditions.

Side Lighting Enhances Morphophysiology by Inducing More Branching and Flowering in Chrysanthemum Grown in Controlled Environment.

Light is one of the most important factors that influence plant growth and development. This study was conducted to examine how lighting direction affects plant morphophysiology by investigating plant growth parameters, leaf anatomy, epidermal cell elongation, stomatal properties, chloroplast arrangement, and physiological changes. In closed-type plant factory units, the rooted cuttings of two chrysanthemum (Chrysanthemum morifolium Ramat.) cultivars, 'Gaya Glory' and 'Pearl Egg', were subjected to a 10 h photoperiod with a 300 µmol∙m-2∙s-1 photosynthetic photon flux density (PPFD) provided by light-emitting diodes (LEDs) from three directions relative to the plant including the top, side, and bottom. Compared to the top or bottom lighting, the side lighting greatly enhanced the plant growth, improved the leaf internal structure and chloroplast arrangement, induced small stomata with a higher density, and promoted stomatal opening, which is associated with an increased stomatal conductance and photosynthetic efficiency. It is worth noting that the side lighting significantly enhanced the induction of branching and flowering for both cultivars., The plants grown with side lighting consistently exhibited the greatest physiological performance. We conclude that the lighting direction had a profound effect on the morphophysiological characteristics of chrysanthemum, and that side lighting dramatically promoted their growth and development, especially in their branching and flowering.

A Novel Lateral Organ Boundary-domain Factor CmLBD2 Positively Regulates Pollen Development by Activating CmACOS5 in Chrysanthemum morifolium.

Male sterility, as a common reproductive characteristic in plants, plays an important role in breeding, in which pollen abortion is a key factor leading to male sterility. Here, based on a low expression level gene CmACOS5 in transcriptome of pollen abortive chrysanthemum, a new transcription factor CmLBD2 of the Lateral Organ Boundaries Domain family, which could bind the promoter of CmACOS5 by yeast one-hybrid library was screened. This study revealed the origin and expression pattern of CmLBD2 in chrysanthemum and verified the functions of two genes in pollen development by transgenic means. Inhibiting the expression of CmACOS5 or CmLBD2 can lead to a large reduction in pollen and even abortion in chrysanthemum. Using yeast one-/two-hybrid, electrophoretic mobility shift assays, and luciferase reporter assays, it was verified that CmLBD2 directly binds to the promoter of CmACOS5. These results suggest that LBD2 is a novel, key transcription factor regulating pollen development. This result will provide a new research background for enriching the function of LBD family proteins and also lay a new foundation for the breeding of male sterile lines and the mechanism of pollen development.

Identification of Chlorophyll Metabolism- and Photosynthesis-Related Genes Regulating Green Flower Color in Chrysanthemum by Integrative Transcriptome and Weighted Correlation Network Analyses.

Green chrysanthemums are difficult to breed but have high commercial value. The molecular basis for the green petal color in chrysanthemum is not fully understood. This was investigated in the present study by RNA sequencing analysis of white and green ray florets collected at three stages of flower development from the F1 progeny of the cross between Chrysanthemum × morifolium "Lüdingdang" with green-petaled flowers and Chrysanthemum vistitum with white-petaled flowers. The chlorophyll content was higher and chloroplast degradation was slower in green pools than in white pools at each developmental stage. Transcriptome analysis revealed that genes that were differentially expressed between the two pools were enriched in pathways related to chlorophyll metabolism and photosynthesis. We identified the transcription factor genes CmCOLa, CmCOLb, CmERF, and CmbHLH as regulators of the green flower color in chrysanthemum by differential expression analysis and weighted gene co-expression network analysis. These findings can guide future efforts to improve the color palette of chrysanthemum flowers through genetic engineering.

New insights into the role of MADS-box transcription factor gene CmANR1 on root and shoot development in chrysanthemum (Chrysanthemum morifolium).

BACKGROUND: MADS-box transcription factors (TFs) are the key regulators of multiple developmental processes in plants; among them, a chrysanthemum MADS-box TF CmANR1 has been isolated and described as functioning in root development in response to high nitrate concentration signals. However, how CmANR1 affects root and shoot development remains unclear. RESULTS: We report that CmANR1 plays a positive role in root system development in chrysanthemum throughout the developmental stages of in vitro tissue cultures. Metabolomics combined with transcriptomics assays show that CmANR1 promotes robust root system development by facilitating nitrate assimilation, and influencing the metabolic pathways of amino acid, glycolysis, and the tricarboxylic acid cycle (TCA) cycle. Also, we found that the expression levels of TFs associated with the nitrate signaling pathways, such as AGL8, AGL21, and LBD29, are significantly up-regulated in CmANR1-transgenic plants relative to the wild-type (WT) control; by contrast, the expression levels of RHD3-LIKE, LBD37, and GATA23 were significantly down-regulated. These results suggest that these nitrate signaling associated TFs are involved in CmANR1-modulated control of root development. In addition, CmANR1 also acts as a positive regulator to control shoot growth and development. CONCLUSIONS: These findings provide potential mechanisms of MADS-box TF CmANR1 modulation of root and shoot development, which occurs by regulating a series of nitrate signaling associated TFs, and influencing the metabolic pathways of amino acid and glycolysis, as well as TCA cycle and nitrate assimilation.

Two Cyc2CL transcripts (Cyc2CL-1 and Cyc2CL-2) may play key roles in the petal and stamen development of ray florets in chrysanthemum.

BACKGROUND: Chrysanthemum morifolium is one of the most popular ornamental crops. The capitulum, which is the main ornamental part of chrysanthemum plants, consists of ligulate marginal ray florets, an attractive corolla (petals), and radially hermaphroditic disc florets, but no stamens. In Asteraceae species, the zygomorphic ray florets evolved from the actinomorphic disc florets. During this process, the zygomorphic ligulate corolla arose and the stamens were aborted. Although molecular genetic research has clarified ray floret development to some extent, the precise molecular mechanism underlying ray floret development in chrysanthemum remained unclear. RESULTS: A CYC2-like gene, Cyc2CL, was cloned from C. morifolium 'Fenditan'. Subsequent analyses revealed that the alternative splicing of Cyc2CL, which occurred in the flower differentiation stage, resulted in the production of Cyc2CL-1 and Cyc2CL-2 in the apical buds. Prior to this stage, only Cyc2CL-1 was produced in the apical buds. A fluorescence in situ hybridization analysis of labeled Cyc2CL-1 and Cyc2CL-2 RNA indicated that Cyc2CL-2 was first expressed in the involucre tissue during the final involucre differentiation stage, but was subsequently expressed in the receptacle and floret primordia as the floral bud differentiation stage progressed. Moreover, Cyc2CL-2 was highly expressed in the inflorescence tissue during the corolla formation stage, and the expression remained high until the end of the floral bud differentiation stage. Furthermore, the overexpression of Cyc2CL-1 and Cyc2CL-2 in transgenic Arabidopsis inhibited stamen and petal development. Therefore, both Cyc2CL-1 and Cyc2CL-2 encode candidate regulators of petal development and stamen abortion and are important for the ray floret development in chrysanthemum. CONCLUSION: In this study, we characterized the alternatively spliced transcripts of the CYC2-like gene that differ subtly regarding expression and function. The data presented herein will be useful for clarifying the regulatory mechanisms associated with the CYC2-like gene and may also be important for identifying the key genes and molecular mechanisms controlling the development of ray florets in chrysanthemum.

Cloning and Functional Characterization of Dihydroflavonol 4-Reductase Gene Involved in Anthocyanin Biosynthesis of Chrysanthemum.

Dihydroflavonol 4-reductase (DFR) catalyzes a committed step in anthocyanin and proanthocyanidin biosynthesis by reducing dihydroflavonols to leucoanthocyanidins. However, the role of this enzyme in determining flower color in the economically important crop chrysanthemum (Chrysanthemum morifolium Ramat.) is unknown. Here, we isolated cDNAs encoding DFR from two chrysanthemum cultivars, the white-flowered chrysanthemum "OhBlang" (CmDFR-OB) and the red-flowered chrysanthemum "RedMarble" (CmDFR-RM) and identified variations in the C-terminus between the two sequences. An enzyme assay using recombinant proteins revealed that both enzymes catalyzed the reduction of dihydroflavonol substrates, but CmDFR-OB showed significantly reduced DFR activity for dihydrokaempferol (DHK) substrate as compared with CmDFR-RM. Transcript levels of anthocyanin biosynthetic genes were consistent with the anthocyanin contents at different flower developmental stages of both cultivars. The inplanta complementation assay, using Arabidopsis thaliana dfr mutant (tt3-1), revealed that CmDFR-RM, but not CmDFR-OB, transgenes restored defective anthocyanin biosynthesis of this mutant at the seedling stage, as well as proanthocyanidin biosynthesis in the seed. The difference in the flower color of two chrysanthemums can be explained by the C-terminal variation of CmDFR combined with the loss of CmF3H expression during flower development.

Standardized Genetic Transformation Protocol for Chrysanthemum cv. 'Jinba' with TERMINAL FLOWER 1 Homolog CmTFL1a.

Chrysanthemum (Chrysanthemum x morifolium Ramat.) cultivar Jinba is a distinctive short-day chrysanthemum that can be exploited as a model organism for studying the molecular mechanism of flowering. The commercial value of Jinba can be increased in global flower markets by developing its proper regeneration and genetic transformation system. By addressing typical problems associated with Agrobacterium-mediated transformation in chrysanthemum, that is, low transformation efficiency and high cultivar specificity, we designed an efficient, stable transformation system. Here, we identify the features that significantly affect the genetic transformation of Jinba and standardize its transformation protocol by using CmTFL1a as a transgene. The appropriate concentrations of various antibiotics (kanamycin, meropenem and carbenicillin) and growth regulators (6-BA, 2,4-D and NAA) for the genetic transformation were determined to check their effects on in vitro plant regeneration from leaf segments of Jinba; thus, the transformation protocol was standardized through Agrobacterium tumefaciens (EHA105). In addition, the presence of the transgene and its stable expression in CmTFL1a transgenic plants were confirmed by polymerase chain reaction (PCR) analysis. The CmTFL1a transgene constitutively expressed in the transgenic plants was highly expressed in shoot apices as compared to stem and leaves. Overexpression of CmTFL1a led to a delay in transition to the reproductive phase and significantly affected plant morphology. This study will help to understand the biological phenomenon of TFL1 homolog in chrysanthemum. Moreover, our findings can explore innovative possibilities for genetic engineering and breeding of other chrysanthemum cultivars.

Comparison of allelopathic effects of two typical invasive plants: Mikania micrantha and Ipomoea cairica in Hainan island.

Mikania micrantha and Ipomoea cairica are two invasive plants widely distribute and seriously damage in Hainan island. In this study, the leaves extracts of two weeds were collected and determined for their allelopathic potentials on Chrysanthemum coronarium. The phytotoxicity bioassay showed that when the extract concentration was 50 and 100 mg/ml, the inhibited effects of M. micrantha on growth of C. coronarium were greater than by I. cairica. However, when the extract concertation at 400 mg/ml, the opposite inhibited effects were observed. We speculated this phenomenon was caused by different allelopathic compounds. Therefore, using gas chromatography-mass spectrometry, 19 and 23 compounds were identified respectively, benzoic acid and cinnamic acid were the main components in the two leaves extracts, which were selected to carry out the further bioassays. Subsequent bioassay results showed the effects of two allelochemicals on morphological index and chlorophyll content and POD activity were all negative to C. coronarium, whereas the content of MDA and activity of SOD, CAT represented adverse changes. Moreover, the inhibitions by cinnamic acid were generally greater than those by benzoic acid. Thus, the phenolic acids played the most crucial roles in the allelopathic effccts of M. micrantha and I. cairica leaves extracts.

Induction of novel inflorescence traits in Chrysanthemum through 60Co gamma irradiation.

PURPOSE: The novelty in flower color or inflorescence form is recognized as a valuable trait in Chrysanthemum - a potential commercial flower crop with significant worth in global cut flower trade. This study was conducted to irradiate white and orange flowered cultivars of Chrysanthemum with an objective to identify and isolate desirable types representing novelty in flower color and inflorescence form from the irradiated populations. The terminal rooted cuttings of Chrysanthemum exposed to γ-irradiation at 10 or 15 Gy doses were found effective for inducing novel flower color variants in cultivars Thiching Queen and Purnima. The mutant progeny evolved with novel inflorescence traits of these cultivars will enrich the existing germplasm of Chrysanthemum for further utilization in breeding programs. MATERIALS AND METHODS: Two standard type Chrysanthemum cultivars, Thiching Queen and Purnima were exposed to varied doses of γ-rays (0, 5, 10, 15, and 20 Gy) using Cobalt 60 (60Co) as irradiation source for treating rooted cuttings. The irradiated mutant population was evaluated for likely variation in various vegetative and flowering characters compared to non-irradiated (control) plants. RESULTS: In Chrysanthemum cultivars Thiching Queen, seven and 'in Purnima', two flower color variants were isolated from the irradiated populations that were reportedly novel in color and desirable for commercial aspect. The leaf abnormalities were observed in mutant populations exhibiting variation in flower color, shape, and size of leaves. Certain floral abnormalities were also observed in inflorescence that reportedly progressed with increase in dosage of γ-rays irradiation. CONCLUSIONS: This study developed a gamma ray (60Co) induced mutagenesis protocol with potential application to develop novel and desirable mutants in Chrysanthemum.

The flower head of Chrysanthemum morifolium Ramat. (Juhua): A paradigm of flowers serving as Chinese dietary herbal medicine.

ETHNOPHARMACOLOGICAL RELEVANCE: Dietary herbal medicines are widely used for the prevention and treatment of a variety of diseases due to their pharmacological activities in China. Juhua (the flower head of Chrysanthemum morifolium Ramat.), the most representative flower-derived one, which is mainly used for the treatment of respiratory and cardiovascular diseases, shows significant activities, such as antimicrobial, anti-inflammatory, and anticancer, and, neuroprotective, as well as effects on the cardiovascular system. AIMS OF THIS REVIEW: This review aims to provide an overview of the crucial roles of flowers in Chinese dietary herbal medicine, and the pharmaceutical research progress of Juhua (the paradigm of dietary herbal medicine derived from the flower) including its applications in Traditional Chinese medicine and diet, cultivars, phytochemistry, quality control, pharmacology, and toxicity, along with chrysanthemum breeding and biotechnology. METHOD: The information associated with Chinese dietary herbal medicine, flower-derived medicine, dietary flower, and pharmaceutical research of Juhua, was collected from government reports, classic books of Traditional Chinese medicine, the thesis of doctors of philosophy and maters, and database including Pubmed, Scifinder, Web of Science, Google Scholar, China National Knowledge Internet; and others. RESULT: All flower-originated crude medicines recorded in Chinese pharmacopeia and their applications were summarized for the first time in this paper. The edible history and development of flowers in China, the theory of Chinese dietary herbal medicines, as well as flowers serving as dietary herbal medicines, were discussed. Moreover, applications in Traditional Chinese medicine and diet, cultivars, phytochemistry, quality control, pharmacology, and safety evaluation of Juhua, together with chrysanthemum breeding and biotechnology, were summarized in this paper. CONCLUSION: The theory of dietary herbal medicines, which are an important part of the Traditional Chinese medicine system, has a history of thousands of years. Many herbal flowers, serving as dietary herbal medicines, contribute significantly to the prevention and treatment of a variety of diseases for Chinese people. To better benefit human health, more effective supervision practice for dietary herbal medicines is needed. Although various investigations on Juhua have been done, there is a lack of analytical methods for discrimination of cultivar flowers and identification of authenticity. Research on the major compounds with bioactivities, especially those related to its clinical application or healthcare function, as well as their possible mechanize, need be strengthened. More safety evaluation of Juhua should be carried out. The research limitations Juhua is facing exist in all dietary herbal medicine.

Deep tillage combined with biofertilizer following soil fumigation improved chrysanthemum growth by regulating the soil microbiome.

Sustained monoculture often leads to the inhibition of plant growth, the decrease of the soil microbial diversity, and changes in soil microbial community composition, particularly to the accumulation of soil-borne pathogens. In this study, we conducted field experiments to investigate the practical effects of tilling the soil down to a depth of 40 cm (40dp) in combination with dazomet (D) soil fumigation and/or the application of a bio-organic fertilizer (B) on chrysanthemum growth, with a focus on the potential mechanisms underlying the responses of the soil microbiome. The growth indices of chrysanthemum were significantly (p < .05) increased in the DB + 40dp treatment compared to that in other treatments. The weighted and unweighted UniFrac distances in the principal coordinate analysis (PCoA) revealed that soil bacterial and fungal community compositions were separated according to the treatments. The abundance of genera potentially expressing growth promotion, such as Pseudomonas and Bacillus, was increased in the DB + 40dp treatment. In addition, the combined DB + 40dp treatment enhanced the activities of catalase, urease, sucrase, and ß-d-glucosidase, and significantly increased the levels of available nitrogen, phosphorus, and potassium in the soil. The redundancy analysis (RDA) implied that the composition of the microbiome was correlated to soil enzymatic activities and soil potassium availability in the rhizosphere soil of chrysanthemum plants. Our findings suggest that the DB + 40dp treatment is a better strategy for improving chrysanthemum growth and regulating the rhizosphere microbiome in monoculture soils than the methods presently employed by commercial chrysanthemum producers.

[Effects of planting density on yield and quality of Chrysanthemum morifolium].

In this paper, five field density treatments were set up in the field plot experiment, which were 2 500,3 000,5 000,6 660,8 000 plants/mu(1 mu≈667 m~2). The agronomic traits, economic traits, mineral element absorption and the content of effective components of Chrysanthemum morifolium under different densities were studied. The results showed that dense planting could significantly reduce the number of secondary branches of Ch. morifolium and the yield per plant, but significantly increase the population yield of Ch. morifolium. The yield of Ch. morifolium was the highest when the density was 8 000 plants/mu, but the effect of increasing yield would gradually decrease with the increase of planting density. With the increase of planting density, the N, P and Mg elements in flowers firstly increased and then decreased. The N element content in leaves increased gradually, which showed that increasing the planting density within a certain range could increase the absorption of N, P and Mg elements in flowers and leaves of Ch. morifolium. The contents of rutin, chlorogenic acid and 3,5-O-dicaffeoyl quinic acid in Ch. morifolium showed a trend of first increasing and then decreasing with the increase of planting density. When the planting density was 5 500,5 000,3 750 plants/mu, the content of chlorogenic acid, rutin and 3,5-O-dicaffeyl quinic acid had the maximum value. The content of luteolin in Ch. morifolium decreased gradually with the increase of planting density. When the planting density was 7 143 plants/mu, the content of luteolin was the minimum. Considering factors such as yield and active ingredient content, the cultivation density of 5 000 plants/mu(row spacing 40 cm×30 cm) can be selected for standard planting of Ch. morifolium.

Morpho-physiological integrators, transcriptome and coexpression network analyses signify the novel molecular signatures associated with axillary bud in chrysanthemum.

BACKGROUND: Axillary bud is an important agronomic and economic trait in cut chrysanthemum. Bud outgrowth is an intricate process controlled by complex molecular regulatory networks, physio-chemical integrators and environmental stimuli. Temperature is one of the key regulators of bud's fate. However, little is known about the temperature-mediated control of axillary bud at molecular levels in chrysanthemum. A comprehensive study was designed to study the bud outgrowth at normal and elevated temperature in cut chrysanthemum. Leaf morphology, histology, physiological parameters were studied to correlate the leaf activity with bud morphology, sucrose and hormonal regulation and the molecular controllers. RESULTS: Temperature caused differential bud outgrowth along bud positions. Photosynthetic leaf area, physiological indicators and sucrose utilization were changed considerable due to high temperature. Comparative transcriptome analysis identified a significant proportion of bud position-specific genes.Weighted Gene Co-expression Network Analysis (WGCNA) showed that axillary bud control can be delineated by modules of coexpressed genes; especially, MEtan3, MEgreen2 and MEantiquewhite presented group of genes specific to bud length. A comparative analysis between different bud positions in two temperatures revealed the morpho-physiological traits associated with specific modules. Moreover, the transcriptional regulatory networks were configured to identify key determinants of bud outgrowth. Cell division, organogenesis, accumulation of storage compounds and metabolic changes were prominent during the bud emergence. CONCLUSIONS: RNA-seq data coupled with morpho-physiological integrators from three bud positions at two temperature regimes brings a robust source to understand bud outgrowth status influenced by high temperature in cut chrysanthemum. Our results provide helpful information for elucidating the regulatory mechanism of temperature on axillary bud growth in chrysanthemum.

The CmbZIP1 transcription factor of chrysanthemum negatively regulates shoot branching.

The basic region/leucine zipper (bZIP) transcription factors play key roles in regulating diverse biological processes in plants. However, their participation in shoot branching has been rarely reported. Here, we isolated a CmbZIP1 transcription factor gene, a member of the bZIP family, from chrysanthemum. Subcellular localization analysis indicated that CmbZIP1 is a nuclear protein. Tissue-specific expression analysis indicated that CmbZIP1 was principally expressed in apical bud and axillary bud. Expression patterns analysis results showed that CmbZIP1 expression was suppressed in axillary buds in response to decapitation but increased in response to shade. Overexpression of CmbZIP1 in Arabidopsis inhibits its shoot branching. In addition, expression of auxin efflux protein PIN-FORMED 1 (PIN1) and auxin signaling components AUXIN RESISTANT 1/3 (AXR1, AXR3) were significantly up-regulated in overexpressing plants in comparison with wild type plants. Moreover, the transcript expression of BRANCHED 2 (AtBRC2) was also significantly up-regulated in overexpressing plants compared with the wild type. Altogether, these results suggest important and negative roles of CmbZIP1 in shoot branching. Our study extends the understanding of the function of bZIP transcription factors in plants and provides valuable gene resources for improving the architectural traits of ornamental plants.

CmCYC2-like transcription factors may interact with each other or bind to the promoter to regulate floral symmetry development in Chrysanthemum morifolium.

The complex capitulum of Chrysanthemum morifolium is often comprised of bilaterally symmetrical ray florets and radially symmetrical disc florets. The TCP transcription factor clade CYCLOIDEA2 (CYC2) appears to play a vital role in determining floral symmetry and in regulating floral organ development in Asteraceae. Our previous study identified six CmCYC2 genes from chrysanthemum and showed that CmCYC2c participated in the regulation of ray floret identity. However, the functions of other CmCYC2 genes and the underlying molecular mechanism of CmCYC2-mediated floral development regulation in chrysanthemums have not been elucidated. In this study, we analysed the function of CmCYC2 genes by ectopic expression of CmCYC2 in Arabidopsis. Then, we examined the protein-protein interaction using yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. Finally, we analysed the protein-DNA interaction using yeast one-hybrid (Y1H) and dual-luciferase reporter assays. We found that ectopic expression of CmCYC2 genes in the Arabidopsis tcp1 mutant changed its floral symmetry and flowering time. Y2H and BiFC assays confirmed three pairs of interactions between CmCYC2 proteins, that is, CmCYC2b-CmCYC2d, CmCYC2b-CmCYC2e and CmCYC2c-CmCYC2d, suggesting that heterodimeric complexes may form between CmCYC2 proteins to increase their functional specificity. The results of Y1H and dual-luciferase reporter assays indicate that CmCYC2c can bind to the promoter of ClCYC2f. Our findings provided clues that CmCYC2-like transcription factors may interact with each other or bind to the promoter to regulate floral symmetry development in C. morifolium. KEY MESSAGE: CmCYC2-like transcription factors may interact with each other or bind to the promoter to regulate floral symmetry development in Chrysanthemum morifolium.

Distribution of cell layers in floral organs of chrysanthemum analyzed with periclinal chimeras carrying a transgene encoding fluorescent protein.

KEY MESSAGE: A fluorescent protein visualized distributions of cell layers in floral organs of chrysanthemum using transgenic periclinal chimeras carrying a gene encoding a fluorescent compound. Plant meristems have three cell layers: the outermost layer (L1), the second layer (L2), and the inner layer (L3). The layers are maintained during development but there is limited knowledge of the details of cell layer patterns within floral organs. In this study, we visualized the distributions of cell layers in floral organs of chrysanthemum using periclinal chimeras carrying a gene encoding a fluorescent compound in the L1 or the L2/L3 layers. The L1 layer contributed most of the epidermal cells of organs including the receptacle, petal, anther, filament, style, stigma, and ovule. The transmitting tissue in the pistil and most of the internal area of the ovule were also derived from the L1. In crossing experiments, no progeny of the L1-chimeric plants showed fluorescence, indicating that the germ cells of chrysanthemum are not derived from the L1 layer. Since anthocyanin pigment is present only in the L1-derived epidermal cells of petals, L1-specific gene integration could be used to alter flower color in commercial cultivars, with a reduced risk of transgene flow from the transgenic chrysanthemums to wild relatives.

Structure and ecological function of the soil microbiome affecting plant-soil feedbacks in the presence of a soil-borne pathogen.

Interactions between plants and soil microbes are important for plant growth and resistance. Through plant-soil-feedbacks, growth of a plant is influenced by the previous plant that was growing in the same soil. We performed a plant-soil feedback study with 37 grass, forb and legume species, to condition the soil and then tested the effects of plant-induced changes in soil microbiomes on the growth of the commercially important cut-flower Chrysanthemum in presence and absence of a pathogen. We analysed the fungal and bacterial communities in these soils using next-generation sequencing and examined their relationship with plant growth in inoculated soils with or without the root pathogen, Pythium ultimum. We show that a large part of the soil microbiome is plant species-specific while a smaller part is conserved at the plant family level. We further identified clusters of plant species creating plant growth promoting microbiomes that suppress concomitantly plant pathogens. Especially soil inocula with higher relative abundances of arbuscular mycorrhizal fungi caused positive effects on the Chrysanthemum growth when exposed to the pathogen. We conclude that plants differ greatly in how they influence the soil microbiome and that plant growth and protection against pathogens is associated with a complex soil microbial community.

Modeling and Optimizing Medium Composition for Shoot Regeneration of Chrysanthemum via Radial Basis Function-Non-dominated Sorting Genetic Algorithm-II (RBF-NSGAII).

The aim of the current study was modeling and optimizing medium compositions for shoot proliferation of chrysanthemum, as a case study, through radial basis function- non-dominated sorting genetic algorithm-II (RBF-NSGAII). RBF as one of the artificial neural networks (ANNs) was used for modeling four outputs including proliferation rate (PR), shoot number (SN), shoot length (SL), and basal callus weight (BCW) based on four variables including 6-benzylaminopurine (BAP), indole-3-butyric acid (IBA), phloroglucinol (PG), and sucrose. Afterward, models were linked to the optimization algorithm. Also, sensitivity analysis was applied for evaluating the importance of each input. The R2 correlation values of 0.88, 0.91, 0.97, and 0.76 between observed and predicted data were obtained for PR, SN, SL, and BCW, respectively. According to RBF-NSGAII, optimal PR (98.85%), SN (13.32), SL (4.83 cm), and BCW (0.08 g) can be obtained from a medium containing 2.16 µM BAP, 0.14 µM IBA, 0.29 mM PG, and 87.63 mM sucrose. The results of sensitivity analysis indicated that PR, SN, and SL were more sensitive to BAP, followed by sucrose, PG, and IBA. Finally, the performance of predicted and optimized medium compositions were tested, and results showed that the difference between the validation data and RBF-NSGAII predicted and optimized data were negligible. Generally, RBF-NSGAII can be considered as an efficient computational strategy for modeling and optimizing in vitro organogenesis.

Sugar Transporter, CmSWEET17, Promotes Bud Outgrowth in Chrysanthemum Morifolium.

We previously demonstrated that 20 mM sucrose promotes the upper axillary bud outgrowth in two-node stems of Chrysanthemum morifolium. In this study, we aimed to screen for potential genes involved in this process. Quantitative reverse transcription (qRT)-PCR analysis of sugar-related genes in the upper axillary bud of plants treated with 20 mM sucrose revealed the specific expression of the gene CmSWEET17. Expression of this gene was increased in the bud, as well as the leaves of C. morifolium, following exogenous sucrose treatment. CmSWEET17 was isolated from C. morifolium and a subcellular localization assay confirmed that the protein product was localized in the cell membrane. Overexpression of CmSWEET17 promoted upper axillary bud growth in the two-node stems treatment as compared with the wild-type. In addition, the expression of auxin transporter genes CmAUX1, CmLAX2, CmPIN1, CmPIN2, and CmPIN4 was upregulated in the upper axillary bud of CmSWEET17 overexpression lines, while indole-3-acetic acid content decreased. The results suggest that CmSWEET17 could be involved in the process of sucrose-induced axillary bud outgrowth in C. morifolium, possibly via the auxin transport pathway.