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.

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.

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.

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.