Effects Of ABA And Its Synthetic Inhibitor STD On Pollen Tube Growth Of Camellia Oleifera

Camellia oleifera is the most economically significant species of woody oil tree in China.Previous research has shown that Camellia oleifera is a late-acting selfincompatible tree,and its molecular mechanism is attributed to the initiation of the process of programmed pollen tube cell death(PCD)after self-pollination.This process is related to the accumulation of a high concentration of abscisic acid(ABA)in the pistil.To explore the role of ABA in PCD of Camellia oleifera pollen tubes,this study utilized the national main cultivar ’Huaxin’ as the experimental material.Using the Camellia oleifera in vitro pollen suspension culture system,we examined the effects of ABA and its synthesis inhibitor sodium tungstate dihydrate(STD)on the growth,structure,and endogenous hormones of Camellia oleifera pollen tubes,as well as their regulation mechanisms.A molecular network involving ABA and STD was constructed to investigate their role in regulating pollen emergence and pollen tube development in Camellia oleifera.The aim was to uncover the mechanism by which ABA PCD in Camellia oleifera pollen tubes.The main findings of the study are as follows:1.Structural characteristics of pollen tube growth of Camellia oleifera under ABA and STD treatment.Using ABA and STD on Camellia oleifera pollen germination culture experiment in vitro.The results indicated that 50μM ABA significantly inhibited pollen sprouting and pollen tube development.The germination rate and pollen tube length decreased by 27.86%and 78.33%,respectively,compared to the control group(CK).During the treatment of pollen with STD,the germination rate and length of pollen tube growth showed an initial increase followed by a decrease with increasing STD concentration.At a concentration of 2 mM STD,the pollen sprouting rate and the pollen tube development length reached their maximum values of 72.71%and 1700.92 μm,respectively.The fluorescent labeling technique revealed that ABA caused depolymerization of the microfilament skeleton but did not affect the structure of the microtubule skeleton.ABA also promotes an increase in calcium ion concentration in pollen tubes.At the same time,it has been found that ABA can inhibit the formation of reactive oxygen species(ROS)in pollen tubes.Additionally,ABA caused the degradation of the nuclear DNA in pollen tubes,leading to the occurrence of PCD in the tubes.2.Characteristics of dynamic changes in endogenous hormones of Camellia oleifera pollen tubes under ABA and STD treatments.The content of endogenous hormones in pollen tubes treated with 50 μM ABA and 2 mM STD were detected by enzyme-linked immunosorbent assay.Results showed that ABA treatment significantly reduced the contents of endogenous zeatin riboside(ZR)and methyl jasmonate(MeJA)in pollen tubes.The ZR content decreased to 0.082 μg/g,while the MeJA content decreased to 2.867 pmol/g.At the same time,the content of endogenous jasmonate(JA)was significantly increased to 4.374 pmol/g.The ABA synthesis inhibitor STD reduced the ABA content in the pollen tube of Camellia oleifera to some extent.The content of endogenous ZR,JA,and MeJA exhibited a similar trend to that observed with ABA treatment during STD treatment.Additionally,STD significantly increased the content of gibberellin A3(GA3)and methyl salicylate(MeSA)in the pollen tube.The study indicated that exogenous ABA treatment has complex synergistic and antagonistic effects on the endogenous hormones in the pollen tubes of Camellia oleifera.3.Transcriptome analysis of abscisic acid and STD in regulating pollen tube growth in Camellia oleifera.Through functional annotation of the Camellia oleifera pollen tube transcriptome Unigenes,31,841 Unigenes were annotated to the GO database,10,255 to the KEGG database,31,921 to the COG database,37,404 to the NR database,32,396 to the PFAM database,and 30,588 to the Swiss-Prot database.A total of 37,568 unigenes were annotated.The annotation results cover all aspects of plant growth and development.Meanwhile,253,2,723,and 2,613 differentially expressed genes(DEGs)were identified from three gene sets(PtABA_vs_PtCK,PtSTD_vs_PtCK,and PtABA_vs_PtSTD),respectively.The above results provide a plethora of candidate genes for studying the molecular mechanisms by which ABA affects pollen tube growth in Camellia oleifera.The DEGs related to pollen tube growth regulation by ABA and STD were further analyzed,including plant hormone signaling pathway,transcription factors(TFs),plant MAPK signal transduction,calcium ion(Ca2+)signal transduction,and microfilament skeleton construction.Combined with the cytological structure and endogenous hormone characteristics,a potential signaling pathway model was constructed,in which ABA and STD were involved in the Camellia oleifera pollen tube.9 DEGs were randomly selected for qRT-PCR authentication,and the outcomes were generally consistent with the findings of the RNA-Seq analysis.In summary,this study confirmed the significant regulatory role of ABA in the programmed death of pollen tubes in Camellia oleifera,from both positive and negative perspectives.ABA altered the expression of Ca2+,ROS,and cytoskeleton-related genes in Camellia oleifera pollen tubes by balancing the transcriptional activity of hormone metabolism,synthesis,and response genes upstream.This led to the degradation of nuclear DNA in the pollen tubes,ultimately resulting in programmed apoptosis of the pollen tubes.