Positional Cloning And Functional Study Of Phosphatidylserine Synthase OsPSS/SUI1 In Rice

The elongation of rice uppermost internode plays a decisive role in plant height development. The related gene isolation and underlying molecular mechanism study in uppermost internode elongation is beneficial to create the plant ideotype by means of molecular breeding. Mutation in phosphatidylserine synthase encoding gene(Os PSS/SUI1) cause the uppermost internode shortening. However, in which way of phosphatidylserine synthase and its product phosphatidylserine(PS) exerting the affect on uppermost internode elongation still remains to be clarified. In this study, we clarified Os PSS/SUI1 or PS biological functions associated to plant cell wall formation. Results from our experiment are summarized as follows:1.A dwarf mutant of japonica var. Kitaake with enclosed panicles, reduced seed setting, and slightly increased tiller number was obtained from tissue culture. The dwarf phenotype was attributed to reduced lengths of both panicles and internodes, especially in the uppermost internode Cytohistological analyses showed no distinct uppermost internode in the mutant. Compared to the orderly arrangement and longitudinal elongation of wild-type(WT) cells, internode cells in the mutant were organized loosely, with enlarged intercellular spaces, and no elongated cells appeared in the uppermost internode of the mutant. Transmission electron microscopy revealed that, compared to the longitudinally elongated and well-arranged parenchymal cells of WT plants, parenchymal cells in the mutant displayed irregular shapes and enlarged cell corners. Some parenchymal cells failed to adhere to adjacent cells.2.Immunoelectron microscopy and high-pressure freezing in parenchymal cells from the uppermost internode revealed that, in sui1-4 cells, large amounts of gold labeling were observed in large compartments in the cytoplasm or accumulated as a complex in vacuolated cells. At the milky endosperm stage, although some gold particles were scattered in the middle lamella of the cell wall,most of the periphery of the intercellular space lacked gold labeling. Some pectin-rich clumps appeared in the outermost layer of the cell wall and protruded from the edge of enlarged intercellular spaces in the cell cap.3.On fractional analysis, Os CESA4 mainly appeared in the polyethylene glycol fraction of WT plants, indicating its presence in the PM, while in sui1-4 plants, strong labeling was observed in the dextran fraction, reflecting its presence in endomembranes. Immunoblotting revealed that the majority of sec GFP was detected in culture medium from WT plants, while most sec GFP was detected in protoplasts from sui1-4 plants, along with very low amounts of the protein in the medium from these plants, suggesting sui1-4 is a secretion defect mutant.4.Chemical analysis of cell-wall components using HPLC revealed that, the cellulose content was12% lower in mutant plants than in WT plants. In the cell-wall lysate, the amount of glucose,contributed from noncrystalline cellulose, xyloglucan and mix linkage glucan, was reduced from 65.1mg/g in WT plants to 35.7 mg/g in sui1-4 plants. In contrast, both xylose and galacturonic acid, the main components of Golgi body-originated hemicelluloses and pectin, were enriched in the uppermostinternode of sui1-4 plants. A general secretion defect resulted in the alterations in cell-wall components in sui1-4 mutant plants.5.Map-based cloning demonstrated that the sui1-4 phenotype was due to an A-to-T point mutation that converted a conserved Asp(position 225) to a Val in a putative PSS(designated Os PSS/SUI1). The transgenic plants obtained was capable of complementing the sui1-4 mutation.. RNA interference(RNAi) transgenic plants displayed a phenotype that mimicked the sui1 phenotype, suggesting that reduced Os PSS/SUI1 expression is sufficient to generate the dwarf phenotype.6.Quantitative reverse transcription polymerase chain reaction(q RT-PCR) of WT plants revealed that Os PSS/SUI1 is expressed in various organs, including roots, culms, and leaves, with the highest expression in panicles. In young transgenic seedlings, GUS expression occurred predominately in the elongation zones of roots, root hairs. Four weeks prior to heading, strong GUS staining was found in the basal parts of internodes(divisional and elongating zones) originating from the intercalary meristem,and shoot apices. One week prior to heading, GUS staining shifted to the basal regions of the uppermost internode and the glumes. During the heading stage, GUS expression was observed mainly in the uppermost internode.7.Os PSS/SUI1 was identified as an integral membrane protein. Subcellular localizations showed that Os PSS/SUI1-GFP fluorescence co-localized only with the ER marker HDEL twelve hours after transformation. However, after 36 h of incubation, this ER co-localization weakened, and additional green fluorescence appeared in some unidentified endomembrane compartments. These signals co-localized with the cis-Golgi apparatus as well as with TGN/EE and PM markers. Signal from Os PSS/SUI1-GFP was independent of signal from markers of the prevacuolar compartment and the tonoplast. GFP-Lac C2 localization pattern was similar to that of Os PSS/SUI1-GFP after 36 h, except for GFP-Lac C2 fluorescence was separated from the ER marker.8.Lipid-overlay assay using purified recombinant maltose-binding protein(MBP)-Os Exo70E1 and MBP-Os Exo70 A revealed that PS bound MBP-Os Exo70E1 and MBP-Os Exo70A1. PS content in panicles and the penultimate internode were reduced. Os Exo70E1 expression-reduced plant showed nearly proportional reduction of internodes. Transmission Electron Microscopy of parenchyma cells in the longitudinal elongation or in horizontal expansion showed their irregular shape and larger intercellular space were similar to those in sui1-4.All these results suggested that sui1-4 is a secretion defective mutant. The mutanted Os PSS/SUI1 affected the content of PS. Os PSS/SUI1 controls cell wall formation in rice by regulating exocytosis through PS.