Study On Effects Of ALA On Tip Vesicle Accumulation Of Pear Pollen Tubes And Its Mechanism

In fruit production system,fruit thinning is always required to achieve high-quality,high-yield and stable fruit production.Hand thinning takes long time and intensive labor efforts,and the chemical thinning agents are unstable and have many side-effects.5-aminolevulinic acid(ALA)is an important synthetic precursor of all organic heterocyclic pyrrole compounds in organisms,and involved in the regulation of plant growth and development.ALA is a natural,non-toxic,biodegradable and environment-friendly biological regulator.Previous studies in our laboratory have found that ALA has a thinning effect,but its mechanism is largely unknown.In this paper,the effects of ALA on the tip vesicles of pear pollen tubes were preliminarily explored using light microscopy,transmission electron microscopy,laser confocal scanning microscopy,non-invasive micromeasurement technology(NMT),and quantitative real-time PCR.The main results of the study are as follows.1.Exogenous ALA treatment at 10 mg·L-1,20 mg·L-1,30 mg·L-1 could inhibit pollen germination and pollen tube growth in a dose-dependent manner.The fluorescence intensity of vesicles in control and ALA treated pollen tubes increased with time in 1 min-20 min,but the fluorescence intensity of the former was significantly lower than that of the latter.At the same time,the distribution of ALA-treated pollen tube vesicles was disordered,distributing at both tip and subtip.This result indicated that ALA-affected pollen tubes lose the characteristic of the apical distribution of vesicles in the control,suggesting that ALA may promote vesicle endocytosis or inhibit exocytosis.The tip Ca2+ flux and Ca2+-ATPase related gene expression during pollen tube growth showed that ALA could inhibit the influx of Ca2+and up-regulate the expression of ACA2 and ACA9,and these effects were dependent on intracellular low concentrations of Ca2+.These results indicated that ALA can reduce intracellular Ca2+ concentration by inhibiting Ca2+ influx and increasing Ca2+ efflux.In addition,calcium signaling is also involved in ALA-inhibited pollen germination and disrupted endocytosis and exocytosis.These results indicated that ALA affects pollen tube growth by reducing Ca2+ concentration and then promoted endocytosis and inhibited exocytosis.The observation of the ultrastructure of the pollen tube tips by transmission electron microscopy further demonstrated that calcium deficiency also plays an important role in the ALA-destroyed organelle structures such as endoplasmic reticulum,Golgi apparatus,and mitochondria.2.To investigate the specific signal pathways of ALA,we introduced HO1(heme oxygenase 1)and NO,two important factors in the metabolism of ALA.(1)Hemin,the HO1 inducer,can inhibit pollen germination and pollen tube growth in a dose-dependent manner.Like ALA,it can also cause pollen tube tip vesicle accumulation and wide distribution,and ZnPP(HO1 inhibitor)alleviated the effect of ALA.These results indicated that the inhibitory effect of ALA on pollen germination was dependent on increasing HO 1 activity and HO1-mediated vesicle accumulation.(2)10 mg·L-1-30 mg·L-1 ALA can increase NO content in pollen tubes to varying degrees.SNP at 0.5 mmol·L-1-3.5 mmol·L-1,NO exogenous donor,inhibited the pollen germination and pollen tube growth in a dose-dependent manner.The inhibitory effect of ALA on pollen germination was alleviated by different concentrations of cPTIO(NO scavenger),among which 70 ?mol·L-1 cPTIO's effect was best.T,hese results indicated that ALA can inhibit pollen germination by increasing the intracellular NO content.The staining experiments of FM 4-64 demonstrated that the accumulation of vesicles by ALA is also depended on NO.The upstream and downstream analyses of HO1,NO,and Ca2+,indicated that HO1 was upstream of NO,and NO was upstream of Ca2+.3.Taxol,the microtubule stabilizer,was added to investigate the effect of ALA on the vesicle transport process.The results showed that ALA-inhibited pollen germination was mitigated when the microtubule structure was stabilized by different concentrations of Taxol,and 0.1 mg·L-1 Taxol showed the best effect.ALA can destroy microtubules and cause microtubules to depolymerize.Staining of vesicles demonstrated that the accumulation of vesicles induced by ALA also depends on the vesicles transported by the depolymerized microtubules.The effect of ALA on the vesicle trafficking was explored by adding Taxol.The results showed that the inhibition of ALA on pollen germination was alleviated by different concentrations of Taxol,and 0.1 mg·L-1 concentration showed the best effect.It is shown that ALA can destroy microtubules and cause microtubules to depolymerize;the staining of vesicles concluded that ALA-accumulated vesicles also relied on the vesicle trafficing which was inhibited by the depolymerized microtubules.The effects of ALA on the vesicle tethering and fusion were further investigated from the molecular point of view.ALA could significantly up-regulated the gene expression level of EXO70B1,SEC3A,SEC5A,SEC6,SEC8 and SEC15A in pears,indicating that ALA mainly affected the vesicle tethering process,this effect was dependent on the decreasing Ca2+signal.These results further proved that ALA inhibits exocytosis and disperses distribution of vesicles by reducing Ca2+concentration,thereby inhibiting the process of pollen germination.In summary,the specific mechanism of ALA in inhibiting the pollen germination process is:ALA increases the NO content in pollen tube by increasing HO1 activity,also decreases the concentration of Ca2+,which located downstream of NO,destroys the cell structure of endoplasmic reticulum,Golgi apparatus and mitochondria,and depolymerizes the microtubules.These effects inhibit exocytosis,promote endocytosis of vesicles,and result in accumulation and disperse distribution of vesicles,which eventually inhibits pollen germination and pollen tube growth.