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.

Polyploidy Induction and Morphological Characteristics in the Asiatic Hybrid Lily(Lilium spp.cultivar dazzling)

The Asiatic hybrid lily cultivar Lilium dazzling(family Liliaceae)is a perennial herbaceous bulbous plant with bright yellow flowers of ornamental values.It also shows resistance to diseases and abiotic stress,making it an ideal parent for breeding studies.This study established a sterile culture system using scales of dazzling lily as explants to induce polyploidy.Adventitious buds growing to 1 cm were treated with different concentrations of colchicine(125,250 and 500μmol·L-1),pendimethalin or trifluralin(100,200 and 300μmol·L-1,respectively)for 12,24 and 36 h.Flow cytometry and chromosome counting were used to identify ploidy,and the phenotype of the polyploids was examined by stomatal observation and leaf index determination.With increasing mutagen concentration and exposure time,the mortality and mutagenic rate increased.The optimal treatment group was:300μmol·L-1 trifluralin for 12 h,which resulted in 15.55%mortality rate and 42.22%induction rate.The polyploid plants showed a significant increase in stomata length,leaf length and leaf width,with a decrease in stomatal density compared with the control plants.This study provided a basis for polyploid breeding.

WRKY11 up-regulated dirigent expression to enhance lignin/lignans accumulation in Lilium regale Wilson during response to Fusarium wilt

Lilium are highly economically valuable ornamental plants that are susceptible to Fusarium wilt caused by Fusarium oxysporum.Lilium regale Wilson,a wild lily native to China,is highly resistant to F.oxysporum.In this study,a WRKY transcription factor,WRKY11,was isolated from L.regale,and its function during the interaction between L.regale and F.oxysporum was characterized.The ectopic expression of LrWRKY11 in tobacco increased the resistance to F oxysporum,moreover,the transcriptome sequencing and UHPLC-MS/MS analysis indicated that the methyl salicylate and methyl jasmonate levels rose in LrWRKY11 transgenic tobacco,meanwhile,the expression of lignin/lignans biosynthesis-related genes including a dirigent(DiR)was up-regulated.The lignin/lignans contents in LrWRKY11-transgenic tobacco also significantly increased compared with the wild-type tobacco.In addition,the resistance of L.regale scales in which LrWRKY11 expression was silenced by RNAi evidently decreased,and additionally,the expression of lignin/lignans biosynthesis-related genes including LrDIR1 was significantly suppressed.Therefore,LrDIR1 and its promoter(PLrDIR1)sequence containing the W-box element were isolated from L.regale.The interaction assay indicated that LrWRKY11 specifically bound to the W-box element in PLrDIR1 and activated LrDIR1 expression.Additionally,β-glucuronidase activity in the transgenic tobacco co-expressing LrWRKY11/PLrDIR1-β-glucuronidase was higher than that in transgenic tobacco expressing PLrDIR1-β-glucuronidase alone.Furthermore,the ectopic expression of LrDIR1 in tobacco enhanced the resistance to F.oxysporum and increased the lignin/lignans accumulation.In brief,this study revealed that LrWRKY11 positively regulated L.regale resistance to F.oxysporum through interaction with salicylic acid/jasmonic acid signaling pathways and modulating LrDIR1 expression to accumulate lignin/lignans.

Transcriptomic and physiological analyses identifying Lanzhou lily(Lilium davidii var.unicolor)drought adaptation strategies

Drought stress is the main limiting plant growth factor in arid and semiarid regions.The Lanzhou lily(Lilium davidii var.unicolor)is the only sweet-tasting lily grown in these regions of China that offers highly edible,medicinal,health,and ornamental value.The Tresor lily is an ornamental flower known for its strong resistance.Plants were grown under three different drought intensity treatments,namely,being watered at intervals of 5,15,and 25 d(either throughout the study or during specific growth stages).We measured the biomass,leaf area,photosynthetic response,chlorophyll content(SPAD value),and osmoregulation of both the Lanzhou lily and the Tresor lily(Lilium‘Tresor’).Additionally,we employed RNA sequencing(RNA-Seq)and qRT-PCR to investigate transcriptomic changes of the Lanzhou lily in response to drought stress.Results showed that under drought stress,the decreasing rate in the Lanzhou lily bulb weight was lower than the corresponding Tresor lily bulb rate;the net photosynthetic rate,transpiration rate,and stomatal conductance of the Lanzhou lily were all higher compared to the Tresor lily;osmoregulation constituents,such as glucose,fructose,sucrose,trehalose,and soluble sugar,in the Lanzhou lily were comparatively higher;PYL,NCED,and ERS genes were significantly expressed in the Lanzhou lily.Under moderate drought,the biosynthesis of flavonoids,circadian rhythms,and the tryptophan metabolism pathway of the Lanzhou lily were all significant.Under severe drought stress,fatty acid elongation,photosynthetic antenna protein,plant hormone signal transduction,flavone and flavonol biosynthesis,and the carotenoid biosynthesis pathway were all significant.The Lanzhou lily adapted to drought stress by coordinating its organs and the unique role of its bulb,regulating photosynthesis,increasing osmolyte content,activating circadian rhythms,signal transduction,fatty acid elongation metabolism,and phenylalanine and flavonoid metabolic pathways,which may collectively be the main adaptation strategy and mechanisms used by the Lanzhou lily under drought stress.

Monitoring Xylem Transport in the Stem of Lilium lancifolium Using Fluorescent Dye 5(6)-Carboxyfluorescein Diacetate

The xylem undergoes physiological changes in response to various environmental conditions during the process of plant growth.To understand these physiological changes,it is extremely important to observe the transport of xylem.In this study,the distribution and structure of vascular bundle in Lilium lancifolium were observed using the method of semithin section.Methods for introducing a fluorescent tracer into the xylem of the stems were evaluated.Then,the transport rule of 5(6)-Carboxyfluorescein diacetate(CFDA)in the xylem of the stem of L.lancifolium was studied by fluorescence dye in live cells tracer technology.The results showed that the vascular bundles of L.lancifolium were scattered in the basic tissue,the peripheral vascular bundles were smaller and densely distributed,and the closer to the center,the larger the volume of vascular bundles and the more sparsely distributed.The vascular bundles of L.lancifolium are limited external tenacity vascular bundles,which are composed of phloem and xylem.The most suitable method for CFDA labeling the xylem of isolated stem segments of L.lancifolium was solution soaking for 24 h.The running speed of CF in the isolated stem was 0.3 cm/h,which was consistent with the running speed of the material in the field.CF could be transported between the xylem and parenchyma cells,indicating that the material transport in the xylem could be through the symplastic pathway.The above results laid a foundation for the study of the xylem transport mechanism and the xylem pathogen disease of lily.

Positive regulatory role of R2R3 MYBs in terpene biosynthesis in Lilium‘Siberia’

Terpenoids are the main components contributing to the fragrance of Lilium‘Siberia’,and LiTPS2 plays a critical role in the biosynthesis of monoterpenoids.Although the major terpene synthases in Lilium‘Siberia’have been identified,how these TPS genes are transcriptionally regulated remains elusive in this distinguished flower.This study aimed to identify transcription factors that regulate the terpene synthesis in Lilium,and disclose the related underlying transcriptional regulation mechanism.In this study,we identified three R2R3-MYB TFs—LiMYB1,LiMYB305 and LiMYB330,which were involved in regulating the biosynthesis of terpenes in Lilium‘Siberia’.Quantitative real-time PCR showed spatial and temporal expression patterns consistent with the emission patterns of terpene compounds.When LiMYB1,LiMYB305 and LiMYB330were overexpressed in flowers,the release of some main monoterpenes,such as linalool and ocimene,as well as the expression of TPS genes,especially for LiTPS2,were enhanced.A virus-induced gene silencing(VIGS)assay showed that silencing these three LiMYBs decreased the level of monoterpenes by down-regulating the expression of the TPS genes.The yeast one-hybrid and transient expression assays indicated that all three LiMYBs could bind to and activate the promoter of LiTPS2.Moreover,the yeast two-hybrid assay verified that LiMYB1 could interact with LiMYB308 and LiMYB330,indicating their synergistic roles in the regulation of floral terpene biosynthesis.In general,these results indicated that LiMYB1,LiMYB305,and LiMYB330 might play essential roles in terpene biosynthesis in Lilium and would provide a new perspective for the transcriptional regulation of volatile terpenes in flowers.

Effects of intercropping on rhizosphere soil microorganisms and root exudates of Lanzhou lily(Lilium davidii var.unicolor)

Both yield and quality of Lanzhou lily(Lilium davidii var. unicolor) are seriously affected by continuous cropping. We attempted to understand the effects of intercropping on the obstacles associated with continuous cropping of Lanzhou lily(Lilium davidii var. unicolor). The changes of rhizosphere microbial biomass and diversity in interplanting and monoculturing systems were studied by using the Illumina Hi Seq sequencing technique. The contents and composition of lily root exudates were measured by gas chromatography–mass spectrometer(GC–MS). The intercropping results of Lanzhou lily showed:(1) There was no difference in the composition of the rhizosphere soil microbes at the phylum level, but the relative abundance of the microbes decreased; and the relative abundance of harmful fungi such as Fusarium sp. increased. The relative abundance of Pleosporales sp. and other beneficial bacteria were reduced. After OTU(operational taxonomic unit)clustering, there were some beneficial bacteria, such as Chaetomium sp., in the lily rhizosphere soil in the interplanting system that had not existed in the single-cropping system. We did not find harmful bacteria that had existed in the single-cropping systm in the rhizosphere soil of interplanting system. The above results indicated that the changes of relative abundance of soil fungi and bacteria in lily rhizosphere soil was not conducive to improving the ecological structure of rhizosphere soil microbes. At the same time, the microbial composition change is very complex—beneficial and yet inadequate at the same time.(2) Root exudates provide a matrix for the growth of microorganisms. Combined with the detection of root exudates, the decrease in the composition of the root exudates of the lily was probably the reason for the decrease of the relative abundance of microbes after intercropping. At the same time, the decrease of the relative content of phenolic compounds, which inhibit the growth of microorganisms, did not increase the relative content of rhizosphere soil microorganisms. Changes in amino acids and total sugars may be responsible for the growth of Fusarium sp.. The results showed that the intercropping pattern did not noticeably alleviate the obstacle to continuous cropping of Lanzhou lily, and the change of microbial biomass and diversity was even unfavorable. However, the emergence of some beneficial bacteria, the disappearance of harmful fungi, and other changes with intercropping are in favor of alleviation of obstacles to continuous cropping of Lanzhou lily.

Isolation of LiLFY1 and Its Expression in Lily (Lilium longiflorum Thunb.)

LFY family genes play a conserved role in regulating flowering. In this study, the cDNA of LiLFY1 was isolated with the strategy of RT-PCR in combination with RACE from lily （Lilium longiflorum Thunb.）. LiLFY1 encodes a LFY transcriptional factor. The alignment analysis of the deduced LiLFY1 protein with other known LFY family proteins indicates that LiLFY1 is highly homologous with rice RFL and maize FLL. The result of Southern hybridization showed that there are two copies of LFY family genes in lily. LiLFY1 is expressed in young flower buds and shoot apical meristem （SAM） but not in roots, shoots, mature leaves, and mature floral organs. The cloned LiLFY1 gene may be applied to genetic engineering aiming for regulating the flowering time in lily.

GAMYB transcription factor LoMYB65 from lily plays a vital role in pollen development

Lily(Lilium spp.)is an important horticultural crop,but its use is limited due to serious pollen contamination problems.There are many studies on pollen development in model plants,but few on flower crops such as lilies.Gibberellin(GA)is a large class of hormones and plays an important role in plant vegetative growth and reproductive development.GAMYB is a group of the R2R3-MYB family upregulated by gibberellin,and plays an important role in anther development.Here,we isolated a novel GAMYB,named LoMYB65,from lily,which was closely related to the AtMYB65 and AtMYB33 in Arabidopsis.Fluorescence quantitative PCR results showed that LoMYB65 was mainly expressed in lily anthers.LoMYB65 could be activated by 288μmol·L^(-1)GA3treatment and the LoMYB65 protein was located in the nucleus and cytoplasm,and had transactivation in yeast and tobacco leaf cells.The conserved motif within 226 amino acids of the C-terminal of LoMYB65 contributed to its transactivation.Overexpression of LoMYB65 caused dwarf phenotype,unnormal tapetum development,less seeds of siliques in transgenic Arabidopsis plants,the transgenic plants showed partly male sterile.Simultaneously,silencing of LoMYB65 with VIGS(Virus Induced Gene Silencing)in lily anthers caused unnormal pollen development and reduced the pollen amount.Overexpression of LoMYB65 in Arabidopsis and silencing of LoMYB65 in lily resulted in decreased pollen counts,so we speculate that LoMYB65 may be dose-dependent.Overall,these findings suggest that LoMYB65 may play an important role in anther development and pollen formation in lily.LoMYB65 may provide a useful candidate gene for pollenless breeding of lily.

Molecular cloning,characterization and promoter analysis of LbgCWIN1 and its expression profiles in response to exogenous sucrose during in vitro bulblet initiation in lily

Lily(Lilium spp.) is an important ornamental flower, which is mainly propagated by bulbs. Cell wall invertases(CWINs), which catalyze the irreversibly conversion of sucrose into glucose and fructose in the extracellular space, are key enzymes participating in sucrose allocation in higher plants. Previous studies have shown that CWINs play an essential role in bulblet initiation process in bulbous crops, but the underlying molecular mechanism remains unclear. Here, a CWIN gene of Lilium brownii var. giganteum(Lbg) was identified and amplified from genomic DNA. Quantitative RT-PCR assays revealed that the expression level of LbgCWIN1 was highly upregulated exactly when the endogenous starch degraded in non-sucrose medium during in vitro bulblet initiation in Lbg. Phylogenetic relationship, motif, and domain analysis of LbgCWIN1 protein and CWINs in other plant species showed that all sequences of these CWIN proteins were highly conserved. The promoter sequence of LbgCWIN1 possessed a number of alpha-amylase-, phytohormone-, light-and stress-responsive cis-elements. Meanwhile, β-glucuronidase(GUS) assay showed that the 459 bp upstream fragment from the translational start site displayed maximal promoter activity. These results revealed that LbgCWIN1 might function in the process of in vitro bulblet initiation and be in the response to degradation of endogenous starch.

Genetic Diversity of Five Different Lily(Lilium L.)Species in Lithuania Revealed by ISSR Markers

To study the genetic diversity and structure of lily (Lilium L.), we collected 35 samples from Vytautas Magnus University Kaunas Botanical Garden, and analyzed their mutual Simple Sequence Repeat (ISSR) molecular markers. For genetic analysis of lily we chose the lily 6 markers. ISSR data revealed a relatively high level of genetic diversity at the different levels of the group, with 95% of polymorphic loci, effective number of alleles of 1.21, the average expected heterozygosis of 0.15 and Shannon’s information index of 0.26. ANOVA analysis and UPGMA-dendrogram suggested a hierarchical structure between species.

The Study on Induction and Proliferation of Tube Bulbs in Lilium brownii

With different parts of bulb scale as explants, the proliferation method of Guizhou Lilium brownii were studied with 3% sodium hypochlorite and MS medium with different concentrations of hormones. The results show that it is feasible to disinfect the bulbs of Lilium brownii with 3% sodium hypoehlorite, moreover, the sodium hypochlorite is very cheap and harmless to researchers, experimental materials and environment. MS ＋ NAA 0.3 mg/L ＋ 6-BA 1.5 mg/L is optimum for the induction of bulbs and, the basal part of Lilium brownie is the optimum explants. After culture for 25 d on the same medium, the tube bulbs could be obtained with the characteristics of high propagation coefficient, strong and new roots. The survival rate is over 90% for transplantation of tube bulbs with diameter between 1-2cm. The method developed in the present study can proliferate abundant Lilium brownii seedling in short time.

Comparative Study on the Structural and Moisture Characteristics of Leaf from the Plantlets of Three Types of Ornamental Lilium brownii

[ Objective] The study was to compare the structural and moisture characteristics of leaf from the plantlets of three types of omamental lily（ Lilium brownii）. [ Method ] The paraffin sections of leaves of tested lily varieties were prepared and then observed under microscope, and the stomatal characteristics and moisture characteristics of tested lily varieties were measured. I Resaltl All the three ornamental lily varieties show isobilateral leaf, single layer of epicuticula and lower epidermis, and no obvious differentiation of palisade tissue and spongy tissue; their stomata distribute in lower epidermis, and the guard cells are dumbbell-shaped; all of these matedais present high moisture. For the leaf sick- ness, midrib sickness and mesophyll tissue sickness, the order was determined to be oriental lily 〉 Lilium/ongiflorum 〉 Asian lily; of the three types of ornamental lily, Ulium Iongiflorum has the largest stomatai aperture and Asian lily has the smallest; focusing the water potential and moisture, the turn was Asian lily 〉 oriental lily 〉 Lilium Iong＇fflorum. [ Condusion] The study may facilitate the artificial regulation of the growth conditions of the plantlets of ornamental lily.

Different Explants of Lilium lancifolium Have Different Potential to Differentiate and Regenerate in Tissue Culture

[Objective] The aim was to investigate differences in differentiation and regeneration of the explants from different parts of Lilium lancifolium（Yixing Lily） in tissue culture.[Method] The different parts of scale,leaf and root of Yixing Lily were cultured as explants on MS basic medium supplemented with different concentrations of plant growth regulators,so as to compare their capacity to differentiate and regenerate.[Result] The explants had different potential to differentiate（scale root leaf）.The capacity of different scale parts to differentiate was the lower part middle partupper part;the capacity of different leaf parts to differentiate was the leaf base middle part leaf tip;the capacity of different root parts to differentiate was the root base root tip middle part.[Conclusion] Tissue culture could be well applied in propagation of Yixing Lily.

百合属(Lilium)几种植物花粉形态的观察

应用光学显微镜和扫描电子显微镜对百合属(Lilium)植物13个种花粉的形态、表面纹饰、萌发沟等性状的观察结果表明,百合属植物花粉皆为椭球形,具单萌发沟,网状纹饰,而花粉大小、网脊宽度、网眼直径等性状存在差异。在供试的13个种中,麝香百合系品种罗瑞拉(Longiflorum hybrids cv.Lorina)的花粉最大,有斑百合(L.concolor var.buschianum Baker)的花粉最小。花粉形态特征和超微结构对百合属植物的分类有一定价值。

Study on the Integration of T-DNA into Lilium longiflorum

[ Objective] The aim of this study is to obtain transgenic Lilium longiflorum Thumb. [ Method] A two-step method of explant and the T-DNA integration technique were employed to transform Lilium longiflorum via Agrobacterium mediated method. [ Result] The best infection effect appeared under the OD600 value of Agrobacterium within 0.6 -0.8, the addition of 250 mg/L AS could increase the transformation efficiency. The optimal concentration of G418 for screening is 50 mg/L. Some putative transgenic plants of Lilium longiflorum with resistance to G418 showed positive in PCR, preliminarily proving that T-DNA gene had integrated into the genome of lily. [ Conclusion] The study may lay a foundation for breeding excellent lily varieties through TDNA integration technique.

东方百合(Lilium oritential)组织培养研究

以东方百合"索邦"为研究对象,对东方百合组织培养技术进行研究。结果表明:启始培养所用材料的基部诱导率明显高于上部,鳞茎分化芽的数量明显多于鳞片;MS+0.1 mg.L-1BA+0.1 mg.L-1NAA为最佳鳞茎诱导培养基;MS+0.1 mg.L-1BA+0.3 mg.L-1NAA为最佳鳞茎增殖培养基;用1/2MS+0.1 mg.L-1IBA培养基进行生根培养,取得良好的效果。

催花保鲜剂对百合(Lilium)绿蕾催花保鲜生理的研究

探讨了百合切花绿蕾经两种催花保鲜剂处理后的效果 ,结果表明 ,催花保鲜剂能促进绿蕾提前开花 ,同时对开花后的百合切花有延缓衰老的作用 ,减少了切花在瓶插期间叶绿素与蛋白质含量的降解 ,提高了切花的品质和商品价值。处理 增加了开放率和花蕾长度 ,处理 增加了花瓣长和花径大小 ,同时在试验过程中揭示了百合切花经催花剂处理后的水分生理变化规律 ,筛选出最佳的催花保鲜剂为处理 。

RT-PCR方法同步检测兰州百合(Lilium davidii var.unicolor)两种主要病毒

建立并优化了以18S rRNA为内参照的特异性检测兰州百合(Lilium davidii var.unicolor)两种主要病毒:黄瓜花叶病毒(Cucumber mosaic virus,CMV)和百合无症病毒(Lily symptomless virus,LSV)的三重RT-PCR体系。结果表明,52.5°C的退火温度、0.025 U/μL的Taq DNA聚合酶、0.6 mmol/L的dNTP浓度、4 mmol/L的Mg2+浓度、0.4μmol/L的各引物浓度以及30个循环等是三重PCR体系扩增的最佳条件;同时用该优化体系检测了兰州百合不同取样部位的病毒差异,发现LSV在不同取样部位的特异扩增条带的强度比较一致,而CMV差距相对较大,外鳞片CMV相对含量在整个感病植株中最高。为兰州百合主要病毒检测、脱毒组培快繁提供了技术支撑。

辽宁的4种野生百合(Lilium spp.)的核型研究

利用染色体常规压片的方法,对辽宁的4种野生百合进行了核型研究。结果如下:①有斑百合L.con-colorSalisb.var.buschianum(Lodd.)Baker.2n=4m(2SAT)+4st+16t(2SAT);②细叶百合L.pumilumDC.2n=6sm(2SAT)+12st+6t;③朝鲜百合L.amabilePalib.2n=4m(2SAT)+8st+12t(2SAT);④垂花百合L.cernumKom.2n=2m(2SAT)+2sm(2SAT)+12st+8t。它们不仅在染色体相对长度、臂比和次缢痕数目及其分布有区别,而且核型类型也不同。除了细叶百合的核型为3A型,其余的均为稳定的3B型。