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.

LIWRKY39 is involved in thermotolerance by activating LIMBF1c and interacting with LICaM3 in lily(Lilium longiflorum)

WRKY transcription factors(TFs)are of great importance in plant responses to different abiotic stresses.However,research on their roles in the regulation of thermotolerance remains limited.Here,we investigated the function of LlWRKY39 in the thermotolerance of lily(Lilium longiflorum‘white heaven’).According to multiple alignment analyses,LIWRKY39 is in the WRKY IId subclass and contains a potential calmodulin(CaM)-binding domain.Further analysis has shown that LlCaM3 interacts with LlWRKY39 by binding to its CaM-binding domain,and this interaction depends on Ca^(2).LIWRKY39 was induced by heat stress(HS),and the LIWRKY39-GFP fusion protein was detected in the nucleus.The thermotolerance of lily and Arabidopsis was increased with the ectopic overexpression of LlWRKY39.The expression of heat-related genes AtHSFA1,AtHSFA2,AtMBF1c,AtGolS1,AtDREB2A,AtWRKY39,and AtHSP101 was significantly elevated in transgenic Arabidopsis lines,which might have promoted an increase in thermotolerance.Then,the promoter of LlMBF1c was isolated from lily,and LlWRKY39 was found to bind to the conserved W-box element in its promoter to activate its activity,suggesting that LlWRKY39 is an upstream regulator of LlMBF1c.In addition,a dual-luciferase reporter assay showed that via protein interaction,LlCaM3 negatively affected LlWRKY39 in the transcriptional activation of LlMBF1c,which might be an important feedback regulation pathway to balance the LlWRKY39-mediated heat stress response(HSR).Collectively,these results imply that LlWRKY39 might participate in the HSR as an important regulator through Ca^(2+)-CaM and multiprotein bridging factor pathways.

High-efficiency Agrobacterium-mediated transformation of chrysanthemum via vacuum infiltration of internode

Leaf disc transformation is one of the traditional methods that are now widely used in chrysanthemum with highly economical and ornamental value in world flower production,but it depends on plant genotypes and is time consuming and complicated.In addition,the transformation success rate of this method is low,generally ranging from 0.1%to 6.25%.Therefore,a highly efficient transformation system is needed.In this study,we are the first to establish a high-efficient chrysanthemum Agrobacterium-mediated transformation system via vacuum infiltration.Chrysanthemum stem internode explants were used as research material and CmLEC1 was used as a reporter gene.After approximately 3 months of culture and selection,the positive transgenic plants were obtained.Additionally,the positive probability was about 42%.The transformation efficiency was up to 37.7%,and if the escapes were removed,it was 16%.Furthermore,stable expression of CmLEC1 in transgenic'Yuhualuoying'was confirmed by qRT-PCR analysis.These results suggest that this genetic transformation system via vacuum infiltration of chrysanthemum stem internode is highly efficient and convenient,and much better than traditional leaf disc transformation,and it will play an important role in chrysanthemum transformation and functional genetics research.

The transcription factor CmLEC1 positively regulates the seed-setting rate in hybridization breeding of chrysanthemum

Distant hybridization is widely used to develop crop cultivars,whereas the hybridization process of embryo abortion often severely reduces the sought-after breeding effect.The LEAFY COTYLEDON1(LEC1)gene has been extensively investigated as a central regulator of seed development,but it is far less studied in crop hybridization breeding.Here we investigated the function and regulation mechanism of CmLEC1 from Chrysanthemum morifolium during its seed development in chrysanthemum hybridization.CmLEC1 encodes a nucleic protein and is specifically expressed in embryos.CmLEC1’s overexpression significantly promoted the seed-setting rate of the cross,while the rate was significantly decreased in the amiR-CmLEC1 transgenic chrysanthemum.The RNA-Seq analysis of the developing hybrid embryos revealed that regulatory genes involved in seed development,namely,CmLEA(late embryogenesis abundant protein),CmOLE(oleosin),CmSSP(seed storage protein),and CmEM(embryonic protein),were upregulated in the OE(overexpressing)lines but downregulated in the amiR lines vs.wild-type lines.Future analysis demonstrated that CmLEC1 directly activated CmLEA expression and interacted with CmC3H,and this CmLEC1–CmC3H interaction could enhance the transactivation ability of CmLEC1 for the expression of CmLEA.Further,CmLEC1 was able to induce several other key genes related to embryo development.Taken together,our results show that CmLEC1 plays a positive role in the hybrid embryo development of chrysanthemum plants,which might involve activating CmLEA’s expression and interacting with CmC3H.This may be a new pathway in the LEC1 regulatory network to promote seed development,one perhaps leading to a novel strategy to not only overcome embryo abortion during crop breeding but also increase the seed yield.

Overexpression of lily LlWRKY22 enhances multiple abiotic stress tolerances in transgenic Arabidopsis

In our previous study,a heat-induced differentially expressed WRKY-IIe gene LlWRKY22 is isolated from lily(Lilium longiflorum),which acts as a positive role in thermotolerance,but whether it is involved in other stress responses is unknown.Here,the expression of LlWRKY22 was indicated to be positively influenced by heat,salt,or mannitol treatments,and its promoter activity was also enhanced after heat,salt,or mannitol treatments.In addition,LlWRKY22 responded to ABA treatment,which activated its expression and also increased the promoter activity.Overexpression of LlWRKY22 in Arabidopsis contributed to growth defects and early flowering.Simultaneously,compared with the wild type,the ABA sensitivity in transgenic lines was increased in both the germination stage and late growth stage.Further analysis showed that LlWRKY22 overexpression elevated the thermotolerance of transgenic plants and induced the expression of AtDREB2A,AtDREB2B,AtDREB2C,and AtJUB1.The salt and mannitol tolerances of the overexpression lines were also improved.Overall,our results illustrated that LlWRKY22 is affected by heat,salt,and osmotic stresses,and positively regulates heat,salt,and osmotic tolerances,which reveals that it acts as a generalist character responding to different abiotic stresses.And further to that,the regulatory pathway of LlWRKY22 also involves in ABA signaling.

Screening and functional analysis of potential S genes in Chrysanthemum morifolium

S genes are the key genes that cause plant self-incompatibility,to find out the key S genes and understand molecular mechanism of self-incompatibility in chrysanthemum,the stigmas and anthers at different developmental stages of'Q10-22-2'—a self-incompatible chrysanthemum cultivar,were used for RNA sequencing.After bioinformatics analysis,13 candidate pistil S genes and five candidate pollen S genes were excavated.A potential pistil S gene was cloned and named as CmSRK1.Meanwhile,a potential pollen S gene was cloned and named as CmPCP1.qRT-PCR revealed that CmSRK1 was specifically expressed in mature stigmas,and CmPCP1 was specifically expressed in anthers 3 d before maturation.Subcellular localization showed that both CmSRK1 and CmPCP1 were located in the nucleus and the cell membrane.Transcriptional activation activity analysis indicated that both of the two proteins had no transcriptional activation activity.Yeast two hybrid assay showed that there was no interaction between CmSRK1 and CmPCP1.CmSRK1 was constructed on the expression vector containing stigma-specific promoter,and CmPCP1 was constructed on the expression vector containing pollen-specific promoter,they were then transformed into Arabidopsis thaliana.Artificial hybridization was performed with transgenic lines containing CmSRK1 as the female parents,and transgenic lines containing CmPCP1 as the male parents.The hybridization results showed that seed sets of two transgenic lines were 19.62%and 11.64%,respectively,while cross-pollinated seed sets of Col-0 was 84.43%.Therefore,it was speculated that CmSRK1 and CmPCP1 might be pistil and pollen S genes of chrysanthemum,respectively,and SI of chrysanthemum belonged to SSI.Citation:Wang F,Xu S,Wu Z,Zhong X,Fang W,et al.2021.Screening and functional analysis of potential S genes in Chrysanthemum morifolium.

A LIMYB305‑LlC3H18‑LIWRKY33 module regulates thermotolerance in lily

The CCCH proteins play important roles in plant growth and development,hormone response,pathogen defense and abiotic stress tolerance.However,the knowledge of their roles in thermotolerance are scarce.Here,we identified a heat-inducible CCCH gene LlC3H18 from lily.LIC3H18 was localized in the cytoplasm and nucleus under normal conditions,while it translocated in the cytoplasmic foci and co-located with the markers of two messenger ribonucleoprotein(mRNP)granules,processing bodies(PBs)and stress granules(SGs)under heat stress conditions,and it also exhibited RNA-binding ability.In addition,LIC3H18 exhibited transactivation activity in both yeast and plant cells.In lily and Arabidopsis,overexpression of LIC3H18 damaged their thermotolerances,and silencing of LIC3H18 in lily also impaired its thermotolerance.Similarly,Arabidopsis atc3h18 mutant also showed decreased thermotolerance.These results indicated that the appropriate expression of C3H18 was crucial for establishing thermotolerance.Further analysis found that LIC3H18 directly bound to the promoter of LIWRKY33 and activated its expression.Besides,it was found that LIMYB305 acted as an upstream factor of LlC3H18 and activated its expression.In conclusion,we demonstrated that there may be a LIMYB305-LlC3H18-LIWRKY33 regulatory module in lily that is involved in the establishment of thermotolerance and finely regulates heat stress response.

Cytological and Molecular Characteristics of Pollen Abortion in Lily with Dysplastic Tapetum

Lily was grown worldwide as a fresh cutting flower because of its colorful petals, but its anther contained a large number of pollen grains that cause serious pollen contamination, however, pollen abortion can effectively reduce the level of pollen pollution. Our analysis aims to use cytological observation to detect the critical stage when pollen abortion occurs and to provide comprehensive gene expression information at the transcriptional level. The result showed that pollen abortion in ‘Little Kiss’ began at the mononuclear stage and the callose that covers the microspores failed to degenerate when young pollens were released from the tetrads. In addition, compared with the normally developed one,the tapetum of ‘Little Kiss’ degraded in advance while the degradation of callose was delayed. Furthermore, 103 differentially expressed genes(DEGs) related to the advance degeneration of tapetum cells and callose were found in the expression levels, including 22 transcription factors(TFs). In particular, two β-glucanase genes(endo-1,3(4)-β-glucanase, exo-β-glucanase) responsible for callose degeneration were significantly down-regulated. These results suggested that pollen abortion may occur at mononuclear stage and that early degeneration of tapetum cells resulted in a significant down-regulation of β-glucanase genes. As a result, the callose to cover microspores impedes the formation of pollen walls, which may possibly lead to pollen abortion.