| Dynamic vacuolar changes associated with pollen development,i.e.increase or decrease of volumes through water accumulation or vacuolar fission,are crucial for pollen viability.Mutations at FAB1 A and FAB1 B,genes encoding the kinases converting PI(3,5)P2 from PI3 P,caused male gametophytic lethality due to defective vacuolar fission during pollen mitosis I(PMI),suggesting a key role in dynamic vacuolar organization.However,other genetic components regulating PI(3,5)P2 production and its involvement in pollen germination and tube growth were obscure.We report here the identification and functional characterization of Arabidopsis VAC14 in pollen development and propose that VAC14 functions through positive regulation of PI(3,5)P2.The main results and conclusions presented in this thesis are as follows:(1)Functional loss of Arabidopsis VAC14 resulted in male gametophytic lethalityNo homozygous plants could be obtained from self-pollinated heterozygous plant vac14/+,indicating gametophytic or embryonic lethality.Segregation ratio from reciprocal crosses berween vac14/+ and wild type showed that vac14 was not able to transmit through the male but its female transmission was unaffected.This result suggested that VAC14 is essential for male gametophytic function.(2)Male gametophytic defects of vac14 occurred during pollen mitosis I(PMI)To determine at which stage the development of vac14 pollen started to show defects,we applied semi-thin plastic sections of anthers at different developmental stages.No defects were detected in the pollen sac of vac14/+ anthers before stage 10.However,during PMI when large vacuoles were converted to numerous small ones in wild type,some microspores in the vac14/+ pollen sacs still contained large vacuoles.At later stages,these abnormal microspores aborted in vac14/+.(3)Inhibiting PI(3,5)P2 production mimics pollen developmental defects of vac14To provide evidence that the role of VAC14 during pollen development was through the production of PI(3,5)P2,we took a pharmacological approach by feeding wild-type developing anthers with a PI(3,5)P2-production inhibitor YM201636.Floral buds containing unicellular,bicellular,or tricellular microspores were collected afterwards for optical sectionsby confocal laser scanning microscopy(CLSM).Compared to wild type treated with the solvent DMSO in which microspore development was normal,YM201636 treatment indeed caused bicellular microspores containing enlarged vacuolar structure,and microspores was aborted at tricellular stage,similar to that in vac14/+ anthers.This result supported that the function of VAC14 during pollen development was through positive regulation of PI(3,5)P2production.(4)VAC14 is constitutively expressed and enriched in tapetum and developing microsporeVAC14 is constitutively expressed based on microarrays and by quantitative PCRs.To support the role of VAC14 during pollen development,we generated Pro VAC14:GUS transgenic plants and analyzed its expression pattern by histochemical GUS staining.Indeed,VAC14 was expressed in all cell layers of developing anthers and signals reached the highest level in pollen grains during anther dehiscence.GUS signals were also detected in mature pollen and in vitro germinating pollen tubes,suggesting a role of VAC14 during pollen germination and tube growth.(5)Inhibiting PI(3,5)P2 production or overexpressing SAC1 reduced pollen germination and tube growthTo determine the function of PI(3,5)P2 after pollen maturation,we took a pharmacological approach by applying YM201636 during pollen germination and tube growth.Treatment of pollen grains with YM201636 significantly reduced pollen germination and pollen tube growth compared to those treated with the solvent DMSO.To provide further evidence supporting the role of PI(3,5)P2,we over-expressed a PI(3,5)P2 phosphatase SAC1.As expected,overexpressing SAC1 caused a significant reduction of pollen germination and tube growth,supporting an important role of PI(3,5)P2 in pollen germination and tube growth. |
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