Molecular Cloning And Functional Analysis Of Genes Involved In The Phosphoinositide-Signaling Pathways From Maize

Previous studies have indicated that phosphoinositide-signaling pathways play a key role in plant growth, development and response to environmental stresses. In animal cells, phosphatidylinositol (PtdIns) is one of the most important phospholipids and a major precursor of phosphoinositide-signaling pathways. It is synthesized by phosphatidylinositol synthase (PIS) from CDP-DG and myo-inositol, and then converted to PIP₂ by sequential phosphorylation on D4 and D5 position. PIP₂ can be hydrolyzed by phospholipase C (PLC) into IP₃ and DG, which promotes calcium ion release and activates protein kinase C repectively. However, phosphoinositide-signaling pathways in plants cells are not well understood yet.Maize (Zea mays L.) is an economically important food and forage crop. Its production is ranking the first among all crops in the world. Identifying and isolating agriculturally important genes in maize, and understanding their functions is important for illuminating the molecular mechanism of plant genetics and development, and for elucidating the evolutionary relationship within the cereals.Our research focuses on the molecular characterization and functional analysis of novel PIS gene and PLC gene in maize.Molecular cloning and functional analysis of PIS gene in maizeA cDNA library was constructed from maize seedlings treated with 0.8% NaCl for 24 h. The sequence of OsPIS and AtPIS were served as queries to search GenBank databases with BLASTN program and got several maize ESTs encoding the PIS conserved domain. A pair of primers (PISF1 and PISR1) for RT-PCR was designed and synthesized according to these conserved sequences. A PIS-like cDNA fragment of approximately 0.6 kb was obtained by RT-PCR, and sequence analysis showed that it encoded the PIS conserved domain. The two primers were then used to screen thecDNA library by PCR method. A full-length cDNA that might encode the maize PIS was obtained and named ZmPIS.Sequence analysis showed that this cDNA was 1164 bp long and contained an open reading frame of 648 nucleotides. The predicted protein contained 215 amino acids with a predicted molecular mass of 24 kDa and pi of 8.79. There was an in-frame stop codon upstream to the start codon of this cDNA indicated that the ZmPIS cDNA harbored the complete coding region. Southern blot analysis indicated that ZmPIS was probably a two-copy gene in maize genome. In amino acid level, ZmPIS showed 85%, 63%, 30%, 38% and 29% identity to the PISs from rice, Arabidopsis, rat, yeast and human, respectively. The protein contained D-G-x(2)-A-R-x(8)-G-x(3)-D-x(3)-D consensus that was same as the conserved and characteristic motif of CDP-alcohol phosphotransferase. Besides, the protein contained several phosphorylation sites of protein kinase C (PKC) and Casein kinase II. The ZmPIS cDNA sequence has been deposited into GenBank at the accession number of AY370763.To clarify the function of ZmPIS gene, the ZmPIS coding region was cloned into the yeast expression vector pYES2/NTA (.GALI promoter) and transformed into the yeast pis mutant YPR113W (genotype: Mat a/a;his3Al/his3Al;leu2A0/leu2A0;lys2A0/LYS2;MET15/metl5A0;ura3A0/ura3A0;YPR113w::kanMX4/YPR113w). Transformant spores were randomly picked and analysed. The results indicated that the expression of ZmPIS gene in YPR113W could complement the pis mutant and suppress the lethal phenotype of a-type haploid. Hence, ZmPIS cDNA was able to encode functional PIS.Northern blot analysis showed that the expression of ZmPIS gene was specifically induced by drought, salinity and low temperature. Interestingly, ZmPIS gene was found to be rapidly and transiently induced to the maximum transcript level after 2 h drought treatment. This suggested that ZmPIS may play an important role in signal transduction of dehydration and other environmental stress.To investigate the physiological function of ZmPIS, the full length ZmPIS cDNA driven by 35S promoter in both orientations was transformed into tobaccos.Expression of the transgene was confirmed by Northern blot analysis and RT-PCR analysis. Under normal growth conditions, no phenotypic changes could be observed in both sense and antisense ZmPIS transgenic plants. However, the over-expression of sense ZmPIS gene in transgenic plants displayed an improved drought or salt tolerance compared with wild-type, empty-vector control plants and antisense plants. The improved drought tolerance was demonstrated by lower ion leakage, lower solution potential, higher water content, higher amino acid concentration in the ZmPIS sense leaves. The lower ion leakage and higher amino acid concentration were also observed in ZmPIS sense leaves under salinity.Molecular cloning and functional analysis of PIC gene from maize According to the conserved domains of PI-PLCs from other plants, a pair of primers (PLCF1 and PLCR1) was designed and synthesized for RT-PCR from maize. A PI-PLC-like cDNA fragment of approximately 1 kb was obtained. Sequence analysis showed that the fragment encoded part of the X and Y conserved domain of PI-PLC sequences reported previously. We screened the maize cDNA library using PCR method and plaque hybridization analysis. Several positive clones were obtained and the longest insertion DNA in these clones was chosen for further analysis. This longest cDNA was named ZmPLC.Sequence analysis indicated that the total length of the ZmPLC cDNA was 2072 bp comprising an open reading frame of 1761 nucleotides. The ZmPLC cDNA encoded a protein of 586 amino acid with a calculated molecular-mass of 65.7 kD and pi of 6.4. The amino acid sequence of the predicted protein contained the X, Y and C2 domains that were conserved in various PI-PLCs from other plants. Alignment analysis showed that the ZmPLC gene had high homology to other PLC homologues at nucleotide level as well as amino acid level. At amino acid level, it showed 60%, 59%, 59%, 58%, 56% and 49% identity to tlje PI-PLC from tobacco, rice, and potato, peas, soybeans, and Arabidopsis, respeeijvelY*^Southern blot analysis,indicated that there were at least two ZmPLC gene copys< in maize genome. Northern blot analysis showed that the expression of the ZmPLC gene in root was up-regulated under salinity, drought, and chUling'Sfress. These results suggested that the Zmg&Gg/sm played aaimportant role in the signal transduction under stress.- -'?â– â–  The yeast plcl nwrtantiYPL268W (genotype* Mat a;his3Al;leu2&0;lys2A0;ura3A0;YPL268w::kariMX4) were viable butdispfayed defects in the utilization of galactose, raffinose, and giycerol at permissive temperatures (23 "C to 30 -X2). The coding region of ZmPLC cDNA was cloned into the yeast expression vector pYES2/NTA (GAL1 promoter) and transformed into YPL268W. Growth rate analysis on medium containing galactose proved that the expression of the ZmPLC gene in YPL268W could accelerate the growth rate of plcl mutant, which suggested that the ZmPLC gene encoded a functional PLC. Recombinant ZmPLC protein was expressed in Escherichia coli. In addition, polyclonal antibody of ZmPLC was obtained, which would be important for further studies to assess the ultimate function of the ZmPLC gene in plants.Transgenic tobacco plants expressing ZmPLC gene in sense and antisense orientations were obtained and analyzed. The results showed that the over-expression of ZmPLC gene in transgenic plants could lead to tolerance to drought stress in contrast to wild-type, empty-vector control plants and ZmPLC antisense plants. Under salt stress conditions, lower ion leakage and higher amino acid concentration were detected in ZmPLC sense transgenic tobacco leaves than in wild-type, empty-vector control ones and ZmPLC antisense ones, which suggested an important role of ZmPLC in conferring salt tolerance to plants.Since transgenic tobacco with the empty-vector alone did not change its resistance to drought or salt stress, we concluded that the improvement of drought or salt tolerance was caused by the over-expression of the ZmPIS or ZmPLC in transgenic tobacco. These observations suggested that both ZmPIS and ZmPLC genes were key factors in signal transduction events under stress. This proposed interrelationship should provide a starting point for further research on the roles of PI systems in plant signaling and metabolic pathways.More studies are needed to elucidate the detailed mechanisms of ZmPIS or ZmPLC gene in die plant responses to environmental stress. Using the transgenic plants and molecular biology techniques as well as biochemistry and physiologicaltechniques, it is possible to illuminate the functions of PIS and PLC genes in plant signal transduction pathways and signal amplification.