Cloning And Expression Of PIPs Gene In Populus And Aquaporins Role In Embolism Recovery

Water is one of the five key factors of affecting plant growth, and it can be thought that alarge amount of water is necessary for plant growth and development. The effect of xylemembolism is the most directly to block the water transport. Water channel proteins in themembrane are involved in controlling the speed of water flux across cellular membranes andwater mass flow intercellular. Long-distance water transport in plants and water uptake of plantroot by casparian strip might invovle water mass flow. Although many scholars have proposedwater channel proteins may be involved in xylem embolism repair, but its mechanism is rarelyreported.In this study, we firstly explored the possible mechanism of xylem embolism repair in the84K poplar(Populus alba and P. glandulosa). And then, we isolated the full length cDNAclones encoding PtPIP1;1, PtPIP1;3, PtPIP1;5, PtPIP2;1, PtPIP2;2, PtPIP2;3, PtPIP2;4,PtPIP2;7and PtPIP2;8from parenchyma cells surrounding xylem of the84K poplar andstudied their expression patterns in the drought stress-rewatering process and in differentorgans using relative real-time PCR. Meanwhile, in order to validate the function of genes,13plant expression vectors of the five genes were constructed and transformed into84K poplarand Arabidopsis thaliana, respectively. The main results were as follows:1. Dynamics in root pressure and PIPs differential expression along the stem werecoincided with changes in the PLC, suggesting that root pressure and PIPs integrated to refillthe embolized vessels, respectively, from basal to top and from top to basal.2. The84K poplar is a "cavitation fatigue"species. It is more sensitive to temperature andhumidity after drought stress, and more vulnerable to xylem cavitation.3. The discrimination between control and drought-stressed leaves is clearer usingfluorescence data than using photosynthesis data. After relatively short durations of severedrought, maximum quantum efficiency of PSII (Fv/Fm) was not significantly affected by thedrought treatment and remained around0.8throughout all experiments, which is in accord with previous reports that the functionality of the photosynthetic apparatus seems to be preserved.The non-photochemical quenching parameter, NPQ, significantly increased in D10stage,suggesting a photoprotective mechanism in leaves. In short, no irreversible damages tophotosynthetic reaction centers occurred throughout this experiment.4. In this study, we selected10reference gene candidates, and examined their expressionprofiles of parenchyma cells surrounding xylem of84K poplar in the drought stress-rewateringprocess and in different organs. Three analytical software packages (GeNorm, NormFinder,and Bestkeeper) were used to assess the stability of gene expression. The results show thatEIF-5A, EF4B-L and UBQ are the optimum pair of reference genes in the droughtstress-rewatering process and EIF-5A, CYP, and UBQ are the more stable gene across differenttissues.5. Using various bioinformatics tools, we predicted basic physical properties,hydrophobicity, transmembrane regions, function and prosite motifs for PtPIP1;1, PtPIP1;3,PtPIP2;3, PtPIP2;7and PtPIP2;8. They all possess the MIP family signal consensus sequenceand the highly conservative sequence of the higher plant, which are the typical structure ofplasma intrinsic proteins, and exhibit six transmembrane domains and the basic symmetricdistribution. The results show that PtPIP1;1, PtPIP1;3, PtPIP2;3, PtPIP2;7and PtPIP2;8arestable hydrophobic proteins, containing the six transmembrane domains and five loops. Itshould be pointed out that N-terminal and C-Terminal of PtPIP2;7and PtPIP2;8are not insidecytoplasm. In addition, C-Terminal of PtPIP1;3contains one micro-positioning signals andPtPIP2;8(285-287) contains one glycosylation sites. According to the results of bioinformaticsanalyses, we choosed PtPIP1;3, PtPIP2;7and PtPIP2;8.6. The expressions of these five genes were detected in different organs by using relativereal-time PCR technology. The results show that different genes had different expressionpatterns and tissue specificity and they are as follows: parenchyma cell surrounding xylem ofthe84K poplar and Populus tomentosa, PIP1;3> PIP2;3> PIP2;2> PIP2;1and PIP1;3>PIP2;3> PIP2;2> PIP2;1, respectively, and the root of the84K poplar, PIP2;8> PIP1;3>PIP1;1> PIP2;7. 7.13plant expression vectors of these five genes(PtPIP1;1, PtPIP1;3, PtPIP2;3,PtPIP2;7and PtPIP2;8)were constructed and transformed into Arabidopsis thaliana and84Kpoplar via Agrobacterium tumefaciens-mediated flower-dipping and leaf disk transformationmethod, respectively.8. Transformed Arabidopsis thaliana of over expression vectors of PtPIP1;1, PtPIP2;3,and PtPIP2;8were obtained after genomic PCR detection. Compared with wild-typeArabidopsis thaliana, over expression of PtPIP1;1in Arabidopsis thaliana. showed more rapidgrowth, higher the transpiration rate, bigger stomatal density, higher leaf relative water content,smaller leaf palisade cells and stomatas. We thought that the over expression of PtPIP1;1might cause cell wall integrity signaling trigering, and then cause the change of cell wallbiosynthesis.9. The transgenic84K poplar of13plant expression vectors of these five genes (PtPIP1;1,PtPIP1;3, PtPIP2;3, PtPIP2;7and PtPIP2;8) were obtained after genomic PCR detection. Forthe moment, We have obtained a lot of cuttings of all transgenic84K poplar.