A Bi-Functional Xyloglucan Galactosyltransferase Is an Indispensable Salt Stress Tolerance Determinant in Arabidopsis

Salinity is an abiotic stress that substantially limits crop production worldwide. To identify salt stress tolerance determinants, we screened for Arabidopsis mutants that are hypersensitive to salt stress and designated these mutants as short root in salt medium （rsa）. One of these mutants, rsa3-1, is hypersensitive to NaCI and LiCI but not to CsCI or to general osmotic stress. Reactive oxygen species （ROS） over-accumulate in rsa3-1 plants under salt stress. Gene expression profiling with Affymetrix microarray analysis revealed that RSA3 controls expression of many genes including genes encoding proteins for ROS detoxification under salt stress. Map-based cloning showed that RSA3 encodes a xyloglucan galactosyltransferase, which is allelic to a gene previously named MUR3/KAM1. The RSA3/ MUR3/KAMl-encoded xylogluscan galactosyltransferase regulates actin microfilament organization （and thereby con- tributes to endomembrane distribution） and is also involved in cell wall biosynthesis. In rsa3-1, actin cannot assemble and form bundles as it does in the wild-type but instead aggregates in the cytoplasm. Furthermore, addition of phal- Ioidin, which prevents actin depolymerization, can rescue salt hypersensitivity of rsa3-1. Together, these results sug- gest that RSA3/MUR3/KAM1 along with other cell wall-associated proteins plays a critical role in salt stress tolerance by maintaining the proper organization of actin microfilaments in order to minimize damage caused by excessive ROS.

Adenovirus-mediated expression of pig α(1,3) galactosyltransferase reconstructs Gal α(1,3) Gal epitope on the surface of human tumor cells

Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. Human has no gal epitope due to the inactivation of alpha(1, 3) GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal alpha(1, 3) Gal structures specifically. In this study, a replication-deficient recombinant adenoviral vector Ad5sGT containing pig alpha(1, 3) GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig alpha(1, 3) GT gene into human tumor cells such as malignant melanoma A375, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.

Fusion of EGFP and porcine α 1,3GT genes decrease GFP expression

Objective:To investigate the effect of fusion proteins expressed by the fused gene of porcineα1,3 galactosyltransierase(α1,3 GT) and enhanced green fluorescent protein(EGFP) on the green fluorescence intensity of EGFP.Methods:The fragment containingα1.3GT was firstly recovered after the pcDNA3.1-α1.3GT recombinant vector were digested with BamHl and EcoRI,and then,the resultant fragment was ligated to the pEGFP-N1 vector which was also digested with the same enzymes.The new recombinant eukaryotic expression pEGFP/a 1,3GT vector was obtained and sequenced.The pEGFP/α1,3GT was used to transfect human lung carcinoma cells A549 and HEKC 293FT,and the expression of EGFP was quantitatively analyzed by fluorescent microscope and flow cytometry.Results:The positive percentage of A549 was 80.5%,and that of 293 FT was 86.5%48 hours after the two cell lines both were transfected by pEGFP-N1.The positive percentage of A549 was 75.8%,and that of 293 FT was 81.2%48 hours after the two cell lines were transfected by pEGFP/α1.3GT.The mean fluorescence intensities of A549 transfected with pEGFP-N1 and pEGFP/α1.3GT were 1.21 and 0.956,respectively when compared with that of A549 without transfection.Meanwhile,the those of the 293FT that were transfected with pEGFP-N1 and pEGFP/αl,3GT were 7.66 and 1.00.respectively when compared with that of 293FT cells without transfection.Conclusions:These results suggested that the expression of EGFP gene fused with porcineα1,3GT gene was partly inhibited.

Regulatory Effect of Electroacupuncture on Ceramide Galactosyltransferase Expression in Sciatic Nerve of Experimental Diabetic Rats

Objective: To observe the change of ceramide galactosyltransferase(CGT) expression in sciatic nerve of experimental diabetic rats after electroacupuncture(EA) treatment. Methods: The rat model of diabetic peripheral neuropathy(DPN) was established with Streptozotocin(STZ). Twenty-eight rats were randomly divided into a normal group, a model group, an EA acupoint group, and an EA non-acupoint group, with 7 rats in each group. Acupuncture on points Shenshu(BL 23) and Zusanli(ST 36) was performed in the EA group using EA electronic device with continuous wave, 2 Hz, 20 min every time, once every other day for 12 times. Acupuncture(on the tip of rat tail) in EA non-acupoint group was performed with the same electronic parameters and time. The model group and the normal group were not given any treatment, except the same fixation as that in the EA acupoint group. After 12 treatments, the albumen and mRNA expressions of CGT of sciatic nerve in rats from each group were measured with real-time polymerase chain reaction(RT-PCR), fluorescence quantitative PCR(FQ-PCR) and immunohistochemical methods. Results: After modeling, the CGT expression was significantly lower than that in the normal group(P<0.01); however, after 12 EA treatments, the CGT positive cells in the EA acupoint group were up-regulated and significantly higher than those in the EA non-acupoint group and the model group(P<0.01). The CGT mRNA expression of the rats after modeling was also higher than that of the normal rats(P<0.01), and the expression in the EA acupoint group was significantly lower than those in the model group and the EA non-acupoint group(P<0.01, P<0.05). Conclusion: EA treatment with acupoints displays some therapeutic or preventive effect in the recovery of DPN via reversing the abnormal expression of CGT mRNA in sciatic nerves of experimental diabetic neuropathy rats, and it is worthy of further study.

Galactosylation of rhamnogalacturonan-Ⅱ for cell wall pectin biosynthesis is critical for root apoplastic iron reallocation in Arabidopsis

Apoplastic iron(Fe)in roots represents an essential Fe storage pool.Reallocation of apoplastic Fe is of great importance to plants experiencing Fe deprivation,but how this reallocation process is regulated remains elusive,likely because of the highly complex cell wall structure and the limited knowledge about cell wall biosynthesis and modulation.Here,we present genetic and biochemical evidence to demonstrate that the Cdi-mediated galactosylation of rhamnogalacturonan-II(RG-II)is required for apoplastic Fe reallocation.Cdi is expressed in roots and up-regulated in response to Fe deficiency.It encodes a putative glycosyltransferase localized to the Golgi apparatus.Biochemical and mass spectrometry assays showed that Cdi catalyzes the transfer of GDP-L-galactose to the terminus of side chain A on RG-II.Disruption of Cdi essentially decreased RG-II dimerization and hence disrupted cell wall formation,as well as the reallocation of apoplastic Fe from roots to shoots.Further transcriptomic,Fourier transform infrared spectroscopy,and Fe desorption kinetic analyses coincidently suggested that Cdi mediates apoplastic Fe reallocation through extensive modulation of cell wall components and consequently the Fe adsorption capacity of the cell wall.Our study provides direct evidence demonstrating a link between cell wall biosynthesis and apoplastic Fe reallocation,thus indicating that the structure of the cell wall is important for efficient usage of the cell wall Fe pool.

Enzymatic synthesis of myricetin 3-O-galactoside through a whole-cell biocatalyst

Objective:Myricetin 3-O-galactoside is an active compound with pharmaceutical potential.The insufficient supply of this compound becomes a bottleneck in the druggability study of myricetin 3-Ogalactoside.Thus,it is necessary to develop a biosynthetic process for myricetin 3-O-galactoside through metabolic engineering.Methods:Two genes OcSUS1 and OcUGE1 encoding sucrose synthase and UDP-glucose 4-epimerase were introduced into BL21(DE3)to reconstruct a UDP-D-galactose(UDP-Gal)biosynthetic pathway in Escherichia coli.The resultant chassis strain was able to produce UDP-Gal.Subsequently,a flavonol 3-O-galactosyltransferase DkFGT gene was transformed into the chassis strain producing UDP-Gal.An artificial pathway for myricetin 3-O-galactoside biosynthesis was thus constructed in E.coli.Results:The obtained engineered strain was demonstrated to be capable of producing myricetin 3-Ogalactoside,reaching 29.7 mg/L.Conclusion:Biosynthesis of myricetin 3-O-galactoside through engineered E.coli could be achieved.This result lays the foundation for the large-scale preparation of myricetin 3-O-galactoside.