Functional Study Of Plant Inositol Polyphosphate Kinase Gene (IPK2)

Inositol polyphosphate kinase （IPK2, also known as IPMK） is a key component for inositol polyphosphate turnover. IPK2 phosphorylates Ins（1,4,5）P3 successively at the D-6 and D-3 positions to generate Ins（1,4,5,6）P4 and Ins（1,3,4,5,6）P5. Notably, IPK2 was originally identified as a transcriptional regulator of gene expression in arginine metabolism in yeast. IPK2 could stabilize the transcriptional complex ArgR-Mcml, which binds to specific promoters possessing the consensus sequences designated "arginine boxes". Recent study by a method for transcriptional assay suggests that this arginine transcriptional response does not require the kinase activity of IPK2. Therefore, IPK2 is a multifunctional protein, both the kinase activity and non-catalytic activity of IPK2 are critical for its function. In addition to roles in the IPs turnover and transcriptional regulation, yeast and mammalian IPK2s have been reported to have phosphatidylinositol 3-kinase （PI3K） activity, which specifically phosphorylates phosphatidylinositol （4,5）-bisphosphate to phosphatidylinositol （3,4, 5）-trisphosphate. Moreover, IPK2 can physically interact with and physiologically regulate target of rapamycin （TOR） and AMP-activated protein kinase （AMPK） in mammals to monitor the cell energy status and coordinate the responses to nutrient availability.The inositol polyphosphate kinase in higher plants was first identified from Arabidopsis thaliana. Genome survey and phylogenetic analysis reveal that there are two highly homologous inositol polyphosphate kinases （AtIPK2a and AtIPK2P） in Arabidopsis. Prior work by overexpressing AtIPK2fβ in Arabidopsis and tobacco revealed its roles in auxiliary shoot branching and abiotic stress responses. However, the endogenous function for AtIPK2 is still unknown. Moreover, whether the kinase activity of IPK2 is involved in specific physiological function or developmental process in plant is completely unknown. In this study, through T-DNA knockout mutant, we systematically investigate the physiological function for AtIPK2a and AtIPK2β in Arabidopsis. We also examined the correlation between the kinase activity and function of AtIPK2. Furthermore, to understand the physiologic function of inositol polyphosphate kinase gene （OsIPK2） in rice, T-DNA insertional mutants for OsIPK2 was searched and identified. In addition, the role of OsIPK2 in plant development and stress response was investigated through heterologous expression in Arabidopsis. The main results of this work are as follows:1. Lethality of atipk2aatipk2β double mutant. Since no visible phenotype was observed for atipk2a and atipk2β single mutant, crosses between atipk2a and atipk2β were performed to generate double mutant. Of over 200 F2 plants, no homozygous double mutant was identified. From the F2 population, we identified the lines of atipk2a/atipk2a;AtIPK2β/atipk2β and AtIPK2a/atipk2a;atipk2β/atipk2βplants. Once again no homozygous double mutant could be obtained from their self-fertilized progeny, supporting the lethality of atipk2aatipk2β double mutant.2. Inhibited male gametophyte transmission of atipk2aatipk2β double mutant. When atipk2a/atipk2a;AtIPK2β/atipk2β or AtIPK2a/atipk2a;atipk2β/atipk2β was pollinated with wild-type pollen, approximately 50% progeny contained double mutant alleles. However, when atipk2a/atipk2a;AtIPK2βi/atipk2β or AtIPK2a/atipk2a; atipk2β/atipk2β plants were used as pollen donors, severe reduction in co-transmission of atipk2a and atipk2β was observed. These results suggested that mutation of AtIPK2a and AtIPK2β significantly reduced the genetic transmission of male gametophyte, but did not affect the female gametophyte function. Complementation analysis confirmed that the lethality of atipk2aatipk2β double mutant was caused by T-DNA insertions in AtIPK2 loci.3. Defective pollen development and pollen tube guidance of atipk2aatipk2β double mutant. Male gametophyte transmission defect could be caused by defects in pollen viability, germination, pollen tube growth and/or fertilization. Microscopic investigation revealed that approximately 20% atipk2aatipk2β double mutant pollen grains have compromised viability. Both in vitro and in vivo assays indicated that the germination and tube growth of viable atipk2aatipk2βdouble mutant pollen were competent. However, the directional growth of atipk2aatipk2βdouble mutant pollen tubes towards the ovule was severely disrupted.4. AtIPK2a and AtIPK2β are important for embryogenesis. Seed set analysis revealed that siliques from self-pollinated atipk2alatipk2a;AtIPK2β/atipk2β and AtIPK2a/atipk2a;atipk2β/atipk2β plants had a variety of defective seeds. Consistent with this, when most of the sibling embryos reached the torpedo stage at 6-7 d after pollination, part of the embryos were found to be retarded at the heart or earlier stages within the same silique. Single-embryo genotyping-PCR suggested that, in addition to the lethality of atipk2aatipk2β double mutant, the embryogenesis of atipk2alatipk2a; AtIPK2β/atipk2βand AtIPK2a/atipk2a;atipk2β/atipk2βare also partially aborted.5. Kinase activities of AtIPK2 are required for the male gametophyte function and embryogenesis. Using site-directed mutagenesis, kinase-dead and substrate specificity altered variants for AtIPK2β （designated as AtIPK2β*） were constructed. These AtIPK2β* variants, together with wild-type control AtIPK2β, were introduced into AtIPK2α/atipk2a;atipk2β/atipk2βplants. Microscopic and genetic analysis revealed that only the normal AtIPK2β could rescue the male gametophyte and embryogenesis defects of atipk2aatipk2β double mutant, while AtIPK2β* variants could not.6. Identification of four rice osipk2 T-DNA insertional mutants. Four rice mutants that containing possible T-DNA insertion in OsIPK2 locus was obtained. Genotyping-PCR confirmed the 3’UTR insertion of OsIPK2 in 03Z11CQ55 and 04Z11MC60, however, no phenotype was observed for them. PFG2C40149 was proved to be a false-positive material, as no osipk2 insertion could be detected. PFG₂C40126 harbored a T-DNA insertion in 5’UTR of OsIPK2, and displayed increased tilling and reduced fertility in the field growth. Unfortunately, the phenotype and osipk2 insertion of PFG2C40126 could not co-segregate.7. OsIPK2 affects the development of Arabidopsis plants. As described above, no T-DNA mutant was currently available for OsIPK2. Hence, heterologous expression of OsIPK2 in Arabidopsis was carried out. Phenotype analysis revealed that OsIPK2 overexpressing Arabidopsis had a variety of alterations in growth and development, including inhibited root length, small and distorted rosette leaves and reduced plant size.8. OsIPK2 enhances abiotic stress response in Arabidopsis. RT-PCR analysis indicated that OsIPK2 is a stress-responsive gene, whose expression was strongly induced by ABA and NaCl treatment. Transgenic Arbidopsis that overexpressing OsIPK2 dispalyed enhanced tolerance to salt and drought stresses.Together, our results demonstrated that AtIPK2a and AtIPK2fβact redundantly to regulate pollen development, pollen tube guidance and embryogenesis in a kinase activity-dependent manner. Moreover, the heterologous expression of OsIPK2 in Arabidopsis had an important guiding significance for endogenous function analysis.