The Analysis On AtPLC2 Function In Plant Development

Phospholipase C (PLC) is a phospholipid catalytic enzyme and play central role in various cellular signaling networks. In mammalian, PLC can be activated by heterotrimetic G protein coupled receptors (GPCRs), which hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5-trisphosphate (IP3). Acting as the substrate of PLC, PIP2 is not only a precursor for some membrane phospholipids but also serves as a membrane anchor for many proteins. DAG can bind to the conserved C1 domains of variety enzymes and stimulate their acticities. IP3 is a major regulator for elevating the cytosolic calcium level. Through binding to the calcium channel that is localized in the endoplasmic reticulum (ER) membrane IP3 promotes releasing calcium from the ER lumen to the cytosol. In animal cells, PLCs take part in diverse cellular processes during the life cycle of animals, such as muscle contraction, cell proliferation and differentiation, visual processing, immune response, nervous system development, sperm-egg cell fusion and zygote development, etc. In plant cells, however, the involvement of a PLC in the life cycle of plants is poorly understood. Although several reports demonstrate that PLCs are crucial in the processes of abscisic acid (ABA) signaling, stomatal movement, disease resistance and thermotolerance, the mechanisms regarding to the PLCs'functions are remained largely unclear. Specifically,9 PLC family members are identified in Arabidopsis genome, but the functions of most of the PLC members such as PLC2 are undetermined. Here, we focused on analyzing the role of PLC2 in the developmental processes of plants, in particular, in the vegetative development stage and in the reproductive development stage in Arabidopsis. Through characterizing the phenotypes of loss-of-PLC2-function, the pivotal role of PLC2 in various aspects of plant development is thus determined. Results showed that PLC2 is involved in the regulations of male and female gametophytes development; moreover, it is also involved in the morphogenesis of the vine and vascular tissues of leaves and in the epidermal cells of leaves. Additionally, PLC2 plays essential role for the root including the root hair development. Further analyses identified that, in reproductive tissues, the function of PLC2 is tightly associated with the level of auxin. Through upregulating the expression levels of auxin biosynthetic YUCs genes, elevation of auxin content in the male and female gametophytes could be achieved, if the PLC2 function is completely abolished. Interestingly, in vegetative tissues, the PLC2 function is linked to the auxin transport. Via modulating the polar localizations of auxin transport PIN proteins, the development of leaves and roots could be altered. Overall, major results from this study are summarized in below paragraphs.1. Expression levels of 9 AtPLC family members were quantitatively compared in various tissues through the qRT-PCR (quantitative real time PCR). Results showed that PLC2 is predominantly expressed in all tested Arabidopsis tissues, suggesting its important roles.2. Some of progenies of T-DNA insertion heterozygous mutant plc2/PLC2 showed embryo-lethal. The resulting progenies from the self-pollinated plc2/PLC2 mutant lines were not able to complete embryogenesis, thus, they were determined as the homozygous. Only 4.06% progenies are segregated from the the self-pollinated plc2/PLC2 mutant lines. Therefore, in the siliques of plc2/PLC2 some of ovules were aborted.3. PLC2 affects the formation of floral organ and the development of male gametophytes. The petals and sepals in the flowers of plc2/plc2 homozygous plants were obviously misformed with comparing to those in the wildtype. In addition, shorten filaments and elongated styles were also observed in plc2/plc2 homozygous flowers. Defective development of anthers and pollens were also produced in plc2/plc2 plants. Incomplete degradation of tapetum and aberrant dehiscence of anthers led to aberrance in microspores and/or pollen grains maturation even pollens pollination. Although few pollen grains were produced in plc2/plc2 homozygous plants they were not able to germinate. Further analysis showed that the increased and prolonged DR5:GUS signal was detected in plc2/plc2 homozygous anther, suggesting the correlation of PLC2 function with the auxin content that might influence the male gametophyte development.4. PLC2 affects the female gametophyte development including the embryo sac development.Likewise, the female gametophyte development was totally abolished in the plc2/plc2 homozygous plants.In plc2/plc2, the first mitosis of megaspore was disoriented and lost its polarity, leading to the abolishment of embryo sac formation. Through analyzing the distribution of DR5:GFP in the ovules, it showed that intensified DR5:GFP signal was detective at the micropylar end of an ovule in which the megaspore mother cell was just developed. Thus, it is likely that the increased auxin level might be produced due to the lost of PLC2 function.5. PLC2 is involved in the regulation of expression of YUC family genes in the reproductive tissue. Auxin biosynthesis gene YUC1, YUC2, YUC4, YUC6, and YUC8 were upregulated dramatically in plc2/plc2 plants, which was further confirmed by analyzing the signal of YUC:GUS in the plc2/plc2 background and with the in situ hybridization experiment that is for characterizing the expression pattern of YUC1 in plc2/plc2 plants. Results demonstrated that the elevation of auxin content in the plc2/plc2 plants was resulted by the increased expression levels of YUCs.6. PLC2 modulates the morphogenesis of leaves.In plc2/plc2, cotyledons and rosette leaves were much smaller than those in the wildtype. The meshwork of vines and vascular tissues in leaves of plc2/plc2 plants showed defectively developed, such as that discontinuance in vascular tissue of leaves was obvious. Shorter lobes and reduced number of circularity of pavement cells were showed in the leaves of plc2/plc2. Disappearance of auxin signal in plc2/plc2 cotyledons may imply that PLC2 may be taken part in the auxin transport.7. PLC2 modulates the root growth. In plc2/plc2 shorter primary roots and decreased gravitropism were noticed. The root apical meristem was gradually collapsed in plc2/plc2 seedlings. Through analyzing the distribution of DR5:GUS it suggested that auxin accumulation in the root tip may be decreased during the growth of plc2/plc2 roots. The root hair growth was also inhibited in plc2/plc2 plants, which could be rescued by application of extracellular NAA and NPA but not by addition of IAA, thus, this result provided us another clue of that PLC2 does play pivotal role in the auxin transport.8. PLC2 plays role in PINs polar targeting to the plasma membrane. Inplc2/plc2, the basal-apical polar localization of PIN2 is affected. The increasing in the lateral membrane localization of PIN2 was detected in plc2/plc2, suggesting the role of PLC2 in the PIN protein recycling and membrane targeting.9. PLC2 modulates the calcium regulation in Arabidopsis. Through analyzing the calcium oscillation in root hairs, it showed that that the calcium level was decreased in plc2/plc2. Meanwhile, expression levels of several calcium related genes were obviously changed in plc2/plc2, suggesting that PLC2 may execute its function via downstream calcium signaling regulation.