Chromosome-scale genome assembly of marigold (Tagetes erecta L.):An ornamental plant and feedstock for industrial lutein production

Marigold(Tagetes erecta L., African marigold) is a widely grown ornamental plant and a main source of the carotenoid lutein for the industrial production of pharmaceuticals, food coloring, and feed additives. To gain a deeper understanding of the genetic mechanism of lutein in marigold, a chromosome-scale assembly of the marigold(T. erecta V-01) genome was completed based on Illumina, PacBio, and Hi-C reads. The707.21-Mb assembled genome consisted of 35 834 annotated protein-coding genes, with 97.7% genomic integrity. We anchored 87.8% of the contigs(covering 621.20 Mb) to 12 pseudochromosomes, bringing the scaffold N50 length to 54.15 Mb. Phylogenetic analysis showed that marigold was closely related to the Asteraceae species bitter vine(Mikania micrantha) and sunflower(Helianthus annuus), all three of which originated in the Americas. Marigold diverged from the sunflower clade 23.57 million years ago(MYA) and from M. micrantha 19.59 MYA.Marigold has undergone three whole-genome duplication events, as well as a recent whole-genome duplication event(WGD-2) common to H.annuus and M. micrantha. Marigold gene families were significantly less expanded than those of M. micrantha or H. annuus, and the marigold genome contained significantly fewer interspersed repeats, which might account for its smaller genome. In addition, a range of candidate genes involved in the lutein biosynthetic pathway were identified. The high-quality reference genome obtained in this study provided a valuable genomic resource for studying the evolution of the Asteraceae family and for improving marigold breeding strategies.

R2R3-type Lo MYB21 affects jasmonate-regulated development and dehiscence of anthers in lily(Lilium oriental hybrids)

Lilies are widely cultivated for cut flowers,but their large anthers carry a considerable amount of colored pollen that is dispersed easily.Studying the molecular mechanism of anther development and dehiscence could help solve this problem.LoMYB21,encoding a putative R2R3v-myb avian myeloblastosis viral oncogene homolog(MYB)transcription factor,was identified from oriental lilies(Lilium‘Siberia’).Real-time quantitative PCR analysis showed that LoMYB21 was mainly expressed in the anther,filament and stigma and had high expression during the late stages of lily anther development.LoMYB21 had transactivation activity and was located in the nucleus through yeast one-hybrid assays and transient expression in Nicotiana benthamiana.Suppression of LoMYB21 by virus-induced gene silencing(VIGS)in Lilium‘Siberia’led to anther indehiscence and reduced the expression of genes related to Jasmonate acid(JA)biosynthesis and signal transduction.Induction of LoMYB21 in DEX::LoMYB21 transgenic Arabidopsis caused procumbent inflorescences that became infertile,accompanied by higher expression of JA biosynthetic and signaling genes.These results demonstrated that JA content and signaling were abnormal in silenced lily and transgenic LoMYB21 Arabidopsis,which affected anther development.Our study indicated that LoMYB21 could regulate lily anther dehiscence through JA biosynthesis and signaling during the late stages of anther development.

Genome-Wide Identification and Expression Analysis of the GSK3 Gene Family in Sunflower under Various Abiotic Stresses

Genes in the glycogen synthase kinase 3(GSK3)family are essential in regulating plant response to stressful conditions.This study employed bioinformatics to uncover the GSK3 gene family from the sunflower genome database.The expressions of GSK3 genes in different tissues and stress treatments,such as salt,drought,and cold,were assessed using transcriptome sequencing and quantitative real-time PCR(qRT-PCR).The study results revealed that the 12 GSK3 genes of sunflower,belonging to four classes(Classes I–IV),contained the GSK3 kinase domain and 11–13 exons.The majority of GSK3 genes were highly expressed in the leaf axil and flower,while their expression levels were relatively lower in the leaf.As a result of salt stress,six of the GSK3 genes(HaSK11,HaSK22,HaSK23,HaSK32,HaSK33,and HaSK41)displayed a notable increase in expression,while HaSK14 and HaSK21 experienced a significant decrease.With regard to drought stress,five of the GSK3 genes(HaSK11,HaSK13,HaSK21,HaSK22,and HaSK33)experienced a remarkable rise in expression.When exposed to cold stress,seven of the GSK3 genes(HaSK11,HaSK12,HaSK13,HaSK32,HaSK33,HaSK41,and HaSK42)showed a substantial increase,whereas HaSK21 and HaSK23 had a sharp decline.This research is of great importance in understanding the abiotic resistance mechanism of sunflowers and developing new varieties with improved stress resistance.

RcSPL1-RcTAF15b regulates the flowering time of rose (Rosa chinensis)

Rose(Rosa chinensis),which is an economically valuable floral species worldwide,has three types,namely once-flowering(OF),occasional or re-blooming(OR),and recurrent or continuous flowering(CF).However,the mechanism underlying the effect of the age pathway on the duration of the CF or OF juvenile phase is largely unknown.In this study,we observed that the RcSPL1 transcript levels were substantially upregulated during the floral development period in CF and OF plants.Additionally,accumulation of RcSPL1 protein was controlled by rch-miR156.The ectopic expression of RcSPL1 in Arabidopsis thaliana accelerated the vegetative phase transition and flowering.Furthermore,the transient overexpression of RcSPL1 in rose plants accelerated flowering,whereas silencing of RcSPL1 had the opposite phenotype.Accordingly,the transcription levels of floral meristem identity genes(APETALA1,FRUITFULL,and LEAFY)were significantly affected by the changes in RcSPL1 expression.RcTAF15b protein,which is an autonomous pathway protein,was revealed to interact with RcSPL1.The silencing and overexpression of RcTAF15b in rose plants led to delayed and accelerated flowering,respectively.Collectively,the study findings imply that RcSPL1–RcTAF15b modulates the flowering time of rose plants.

HbNST1 is a positive regulator of the lignin accumulation in strawflower bracts

Strawflower(Helichrysum bracteatum)capitula have papery bracts and thus have the qualities of a naturally dried flower.The involucral bract cells have a secondary cell wall(SCW)of which a crucial component is lignin.Although the constituents of SCWs have been studied extensively in plants,little is known of the mechanism regulating SCW formation,especially lignin biosynthesis in the involucral bracts of strawflower.In this study,a homolog of NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1,designated Hb NST1,was identified as a positive regulator of lignin biosynthesis in strawflower.The transcript level of Hb NST1 was the highest in the involucral bracts.Subcellular localization analysis indicated that Hb NST1 was localized to the nucleus.Overexpression of Hb NST1 in Chrysanthemum indicum promoted the expression of a gene related to lignin biosynthesis,a homolog of cinnamyl alcohol dehydrogenase,designated Ci CAD,suggesting that Hb NST1 was associated with the accumulation of lignin in the SCW of the involucral bracts.Taken together,the results suggested that Hb NST1 positively regulated lignin accumulation in the involucral bracts and mediated the expression of lignin biosynthesis-related genes in strawflower.

The MADS-box gene RhAGL6 plays a master role in regulating the receptacle malformation in rose at low temperature

Low temperature usually results in the developmental deformity of flower organs,immensely affecting the quality of rose flowers.However,it's largely unknown about the regulatory mechanisms activated by low temperature.Here,we used a low temperature-sensitive Rosa hybrida cv.‘Peach Avalanche’to screen a MADS-box gene RhAGL6 via conjoint analysis between RNA sequencing(RNA-seq)and whole-genome bisulfite sequencing(WGBS).Furthermore,we found that low temperature induced the hypermethylation and elevated histone 3 lys-27 trimethylation(H3K27me3)level on the RhAGL6 promoter,leading to decreased RhAGL6 expression.In addition,RhAGL6 silencing resulted in the formation of abnormal receptacles.We also found that the levels of gibberellins(GA3)and abscisic acid(ABA)in the receptacle under low temperature were lower and higher,respectively,than under normal temperature.Promoter activity analysis revealed that GA3 significantly activated RhAGL6 promoter activity,whereas ABA inhibited it.Thus,we propose that RhAGL6 regulates rose receptacle development by integrating epigenetic regulation and phytohormones signaling at low temperature.

Spatial Process of Green Infrastructure Changes Associated with Rapid Urbanization in Shenzhen,China

Through a case study of Shenzhen City,China,this study focused on a quantitative method for analyzing the spatial processes involved in green infrastructure changes associated with rapid urbanization.Based on RS,GIS and SPSS statistics software,the approach includes selection of the square analysis units and representative landscape metrics,quantification of the change types of landscape metrics in all analysis units through two indices and hierarchical cluster analysis of the above analysis units with different landscape metric change types(i.e.spatial attributes).The analyses verify that there is a significant sequence of continuous changes in green infrastructure in Shenzhen.They are the perforation,the segmentation,the fragmentation,the evanescence and the filling-in processes,which have a good spatio-temporal correspondence with urbanization and reflect the synthetic influence of urban planning,government policies and landforms.Compared with other studies on quantifying the spatial pattern,this study provides an alternative probe into linking the spatial pattern to spatial processes and the corresponding ecological processes in the future.These spatio-temporal processes offer many opportunities for identifying,protecting and restoring key elements in an urban green infrastructure network for areas in the early stages of urbanization or for non-urbanized areas.

Investigating the Effectiveness of Road-related Mitigation Measures under Semi-controlled Conditions:A Case Study on Asian Amphibians

Road traffic is the main factor causing the decline in amphibian populations worldwide. The proper design of an amphibian tunnel is one of the most efficient measures to mitigate the negative impacts of road traffic on amphibians. However, no study has investigated the effectiveness of amphibian tunnels under semi-controlled conditions in Asian amphibians. Here, we selected two representative amphibian species, the Chinese brown frog, Rana chensinensis, and the Asiatic toad, Bufo gargarizans, which suffer the most severe road mortality along the roads in Northeast China. We placed experimental arrays of culverts of various sizes(diameters of 1.5, 1, and 0.5 m for circular culverts; side lengths of 1.5, 1, and 0.5 m for box culverts), and substrate type(soil, concrete, and metal) to examine the preferences of both species during the migratory season between May and September in 2016 and 2017. The results revealed that the Chinese brown frog preferred mid-and large-sized culverts as well as soil culverts. We concluded that culverts with a side length ≥ 1 m, lined with soil, and accompanied by a ≥ 0.4 m high guide drift fence and ≤ 45° gradient on the roadside ditch wall would best facilitate road crossings for both species and likely for other amphibian species in Northeast China.

Optimal design of vegetation in residential district with numerical simulation and field experiment

Vegetation plays a key role in improving wind environment of residential districts,and is helpful for creating a comfortable and beautiful living environment.The optimal design of vegetation for wind environment improvement in winter was investigated by carrying out field experiments in Heqingyuan residential area in Beijing,and after that,numerical simulation with SPOTE(simulation platform for outdoor thermal environment) experiments for outdoor thermal environment of vegetation was adopted for comparison.The conclusions were summarized as follows:1) By comparing the experimental data with simulation results,it could be concluded that the wind field simulated was consistent with the actual wind field,and the flow distribution impacted by vegetation could be accurately reflected;2) The wind velocity with vegetation was lower than that without vegetation,and the wind velocity was reduced by 46%;3) By adjusting arrangement and types of vegetation in the regions with excessively large wind velocity,the pedestrian-level wind velocity could be obviously improved through the simulation and comparison.

A detached petal disc assay and virus-induced gene silencing facilitate the study of Botrytis cinerea resistance in rose flowers

Fresh-cut roses(Rosa hybrida)are one of the most important ornamental crops worldwide,with annual trade in the billions of dollars.Gray mold disease caused by the pathogen Botrytis cinerea is the most serious fungal threat to cut roses,causing extensive postharvest losses.In this study,we optimized a detached petal disc assay(DPDA)for artificial B.cinerea inoculation and quantification of disease symptoms in rose petals.Furthermore,as the identification of rose genes involved in B.cinerea resistance could provide useful genetic and genomic resources,we devised a virusinduced gene silencing(VIGS)procedure for the functional analysis of B.cinerea resistance genes in rose petals.We used RhPR10.1 as a reporter of silencing efficiency and found that the rose cultivar‘Samantha’showed the greatest decrease in RhPR10.1 expression among the cultivars tested.To determine whether jasmonic acid and ethylene are required for B.cinerea resistance in rose petals,we used VIGS to silence the expression of RhLOX5 and RhEIN3(encoding a jasmonic acid biosynthesis pathway protein and an ethylene regulatory protein,respectively)and found that petal susceptibility to B.cinerea was affected.Finally,a VIGS screen of B.cinerea-induced rose transcription factors demonstrated the potential benefits of this method for the high-throughput identification of gene function in B.cinerea resistance.Collectively,our data show that the combination of the DPDA and VIGS is a reliable and highthroughput method for studying B.cinerea resistance in rose.

Auxin response and transport during induction of pedicel abscission in tomato

Auxin plays a central role in control of organ abscission,and it is thought that changes in the auxin gradient across the abscission zone are the primary determinant of the onset of abscission.The nature of this gradient,whether in concentration,flow,or perhaps in the response system has not conclusively been determined.We employed a DR5::GUS auxin response reporter system to examine the temporal and spatial distribution of the auxin response activity in response to developmental and environmental cues during pedicel abscission in tomato.In pedicels of young and fully open flowers,auxin response,as indicated by GUS activity,was predominantly detected in the vascular tissues and was almost entirely confined to the abscission zone(AZ)and to the distal portion of the pedicel,with a striking reduction in the proximal tissues below the AZ-a‘step’,rather than a gradient.Following pollination and during early fruit development,auxin response increased substantially throughout the pedicel.Changes in GUS activity following treatments that caused pedicel abscission(flower removal,high temperature,darkness,ethylene,or N-1-naphthylphthalamic acid(NPA)treatment)were relatively minor,with reduced auxin response in the AZ and some reduction above and below it.Expression of genes encoding some auxin efflux carriers(PIN)and influx carriers(AUX⁄LAX)was substantially reduced in the abscission zone of NPA-treated pedicels,and in pedicels stimulated to abscise by flower removal.Our results suggest that changes in auxin flow distribution through the abscission zone are likely more important than the auxin response system in the regulation of abscission.

Isolation and Functional Analysis of the Regulation of Branching by Isopentenyl Transferase Gene CmIPT1 in <i>Chrysanthemum morifolium </i>cv. ‘Jinba’

Today’s chrysanthemums are highly evolved flowering plants and they are considered as one of the most important ornamental cut flowers. In this research an isopentenyl transferase gene named CmIPT1 was isolated from Chrysanthemum morifolium cv. ‘Jinba’ using RACE and RT-PCR methods. The full cDNA sequence of CmIPT1 was 873 bp which encoded a deduced protein of 290 amino acids. It contained GxxGxGKS which is a conserved sequence of the typical domain of IPT family. The phylogenetic tree analysis of CmIPT1 in Chrysanthemum morifolium cv. ‘Jinba’ shows that it has the closest relationship with CcIPT1 in Cynara cardunculus var. scolymus. Expression of CmIPT1 was higher in stems and apex, whereas it was lower in leaves and roots. And the overexpression of CmIPT1 obviously increased the number of rosette branches in Arabidopsis. Here, in our study, we showed that CmIPT1 is a positive regulator of branch development in Chrysanthemum and may play a key role in regulating lateral branch formation of Chrysanthemum plants.

Agrobacterium tumefaciens-mediated transformation of modern rose(Rosa hybrida)using leaf-derived embryogenic callus

Rose(Rosa hybrida)is widely used for cut flowers and as garden plants.Stable and efficient transformation system is required for functional genomics of rose.Here,we established an efficient transformation method for rose using Agrobacterium tumefaciens-mediated transformation of embryogenic callus.Expanding rose leaves were used as explants to induce somatic embryos,which were subjected to transformation with A.tumefaciens strain GV3101 using Green Fluorescence Protein(GFP)as a marker gene.It took about 8 months to generate transgenic shoots from embryogenic callus.PCR,RT-PCR,Southern and Western blotting,as well as stereoscopic fluorescence microscopy analysis demonstrated that GFP transgenes integrated stably into the rose genome.According to our data,a transformation efficiency of up to 6%can be achieved by following this optimized protocol.

The RhHB1/RhLOX4 module affects the dehydration tolerance of rose flowers(Rosa hybrida)by fine-tuning jasmonic acid levels

Phytohormones are key factors in plant responsiveness to abiotic and biotic stresses,and maintaining hormone homeostasis is critically important during stress responses.Cut rose(Rosa hybrida)flowers experience dehydration stress during postharvest handling,and jasmonic acid(JA)levels change as a result of this stress.However,how JA is involved in dehydration tolerance remains unclear.We investigated the functions of the JA-and dehydration-induced RhHB1 gene,which encodes a homeodomain-leucine zipper Iγ-clade transcription factor,in rose flowers.Silencing RhHB1 decreased petal dehydration tolerance and resulted in a persistent increase in JA-Ile content and reduced dehydration tolerance.An elevated JA-Ile level had a detrimental effect on rose petal dehydration tolerance.RhHB1 was shown to lower the transient induction of JA-Ile accumulation in response to dehydration.In addition to transcriptomic data,we obtained evidence that RhHB1 suppresses the expression of the lipoxygenase 4(RhLOX4)gene by directly binding to its promoter both in vivo and in vitro.We propose that increased JA-Ile levels weaken the capacity for osmotic adjustment in petal cells,resulting in reduced dehydration tolerance.In conclusion,a JA feedback loop mediated by an RhHB1/RhLOX4 regulatory module provides dehydration tolerance by fine-tuning bioactive JA levels in dehydrated flowers.

Proteomic Analysis of High Temperature Stress-Responsive Proteins in Chrysanthemum Leaves

Chrysanthemum is one of the most important ornamental flowers in the world,and temperature has a significant influence on its field production.In the present study,differentially expressed proteins were investigated in the leaves of Dendranthema grandiflorum‘Jinba’under high temperature stress using label-free quantitative proteomics techniques.The expressed proteins were comparatively identified and analyzed.A total of 1,463 heat-related,differentially expressed proteins were successfully identified by Liquid Chromatography-tandem Mass Spectrometry(LC-MS/MS),and 1,463 heat-related,differentially expressed proteins were successfully identified by mass spectrometry after a high temperature treatment.Among these,701 proteins were upregulated and 762 proteins were downregulated.The in-depth bioinformatics analysis of these differentially expressed proteins revealed that these were involved in energy metabolism pathways,protein metabolism,and heat shock.In the present study,the investigators determined the changes in the levels of some proteins,and their expression at the protein and molecular levels in chrysanthemum to help reveal the mechanism of heat resistance in chrysanthemum.Furthermore,the present study elucidated some of the proteins correlated to heat resistance in chrysanthemum,and their expression changes at the protein and molecular levels to help reveal the mechanism of heat resistance in this flower species.These results provide a theoretical basis for the selection of new heat resistant varieties of chrysanthemum in the field.

Plant Regeneration from In Vitro Cultured Hypocotyl Explants of Euonymus japonicus Cu zhi

Adventitious shoots were successfully regenerated from hypocotyl explants of in vitro cultures of Euonymus japonicus Cu zhi. Hypocotyl slices were cultured on Murashige and Skoog （MS） and B5 basal medium supplemented with varied concentration of different plant growth-regulators, e.g., α-naphthaleneacetic acid （NAA） and indole-3-butyric acid （IBA） in combination with 6-benzylaminopurine （6-BA） and kinetin. The study showed that shoots could be directly regenerated from hypocotyl explants without the intervening callus phase; MS medium was more suitable for adventitious shoots regeneration. The ability of hypocotyls segments to produce shoots varied depending upon their position on the seedlings. The highest regeneration rate was obtained with hypocotyl segments near to the cotyledon cultured on MS basal medium supplemented with 1.5 mg L^-1 6-BA and 0.05 mg L^-1 NAA （63.64%）. The regenerated shoots were readily elongated on the same medium as used for multiplication and rooted on half-strength MS basal medium supplemented with 1.0 mg L^-1 IBA and 100 mg L^-1 activated carbon. After being transferred to greenhouse conditions, 96% of the plantlets were successfully acclimatized. This regeneration system is applied for genetic transformation now.

Proteomic Analysis of Chrysanthemum Lateral Buds after Removing Apical Dominance Based on Label-Free Technology

Studying the genetic basis and regulatory mechanism of chrysanthemum lateral bud outgrowth is of great significance for reduction the production cost of cut chrysanthemum.To clarify the molecular basis of lateral bud elongation after removal of apical dominance in chrysanthemum,label-free quantification analysis was used to analyze the proteome changes after apical bud removal.Quantitative real-time PCR(qPCR)was used to analyze the changes in the expression of three plant hormone-related genes.A total of 440 differentially expressed proteins were successfully identified at three time points during the lateral bud elongation.The number of differentially expressed proteins in the three stages(24 h/0 h,48 h/0 h,48 h/24 h)were 219,332,and 97,respectively.The difference in expressed proteins in the three comparison stages mainly involves RNA processing and modification;translation,ribosomal structure and biogenesis;Posttranslational modification,protein turnover,and chaperones.Path analysis showed that there was various physiological activities in the process of lateral bud dormancy breaking and elongation,which involved energy metabolism,biosynthesis,signal transduction and stress response in the growth process of lateral buds.qPCR indicated that the expression of cytokinin synthesis related gene was significantly increased after the removal of apical dominance,while the expression of strigolactones synthesis related gene experiences a dramatic fall to promote the development of the lateral buds.However,there was a drop before a slight increase in the expression of the auxin synthesis related gene,which was mainly due to the removal of apical dominance that led to the loss of indoleacetic acid in the main stem.However,with formation of the new apical source,indoleacetic acid can be released again.

A wake-up call: signaling in regulating ornamental geophytes dormancy

Ornamental geophytes are a group of important flowers worldwide.As perennial plants,geophytes go through several rounds of life cycle under seasonal climates.The dormant trait of underground modified organs in geophytes is critical for the process of storage,planting as well as breeding.Although the dormant physiology in geophytes is complex and largely unknown,several advancements have been achieved in this field.Here,we review the knowledge on the role of environmental factors,endogenous hormones,carbohydrates,and epigenetics in the regulation of geophytes dormancy release(GDR).We also discuss dormancy release(DR)methods and their roles in geophytes,including small molecular chemicals and wounding treatments.

Aquaporin,beyond a transporter

Aquaporins(AQPs)are one of the most ancient superfamily proteins,which are essential for maintaining the fluid homeostasis of most organisms against various environments.Here,the latest findings for function of AQPs in cell signal transduction in plants are summarized.We also put forward several issues that still need to be addressed in the future.

An Ethylene-inhibited NF-YC Transcription Factor RhNF-YC9 Regulates Petal Expansion in Rose

The speed of flower opening is closely related to their ornamental period.Ethylene functions as a negative regulator involved in the regulation of the petal expansion process.In this study,we isolated a NF-YC transcription factor gene,RhNF-YC9,fromrose petals.RhNF-YC9 expression was induced at the early stages of flower opening but was inhibited by ethylene treatment.Silencing RhNF-YC9 decreased the speed of petal expansion from stage 2 to stage 5.The expressions of 11 cell expansion-related genes involved in cell wall loosening,cell turgor modulation,and cytoskeleton remodeling were significantly down-regulated in RhNF-YC9-silenced petals.We also found that silencing RhNF-YC9 decreased the expression of gibberellin acid(GA)biosynthetic gene RhGA20ox while significantly increasing the transcripts of GA catabolic gene RhGA2ox,reducing the accumulation of GA4 and GA7.The influence of ethylene treatment on the expression of RhGA20ox and RhGA2ox showed the same trend.These results together suggested that RhNF-YC9 positively regulated the speed of petal expansion and mediated the crosstalk between ethylene and GA.Our findings revealed a new insight into the function of NF-YC transcription factors involved in ethylene-regulated petal expansion.