| Wax form a continuous lipid membrance covering the epidermal cells of all aerial plant organs. It mainly comprised of lony-chain aliphatic compounds derived from very long chain fatty acids that are enlonged from C16- or C18 -long fatty acids in ER by many fatty acid elongation (FAE) complexs. The chemical and physical properties of cuticular waxes indicate that they have vital functions for plant life. The primary role of cuticular waxes is to restrict non-stomatal waterloss. Waxes also have many other functions, such as protection against UV radiation and resisting bacterial and fungal pathogenes. However, the metabolic and regulator- y processes of waxes synthesis are still in enigmatic. Many models have been put out to elucidate these processes of wax biosynthesis and wax secretion. Wax precursors are synthesized in the plastids and enlongated to very long acyl-CoA chains as direct processors in ER. Once the very long acyl-CoA chains are synthesized, they are converted to cuticular waxes by four different pathways. It is not know how cuticular waxes reach the epidermal surface from in- tracellular. The pathway most likely involves endoplastric reticulum, transport vesicles, substrate ligands, vesicle receptors and many other secretory factors. However, more and more evidances provid that nsLTPs may be involved in wax transport to the surface.A group of proteins termed nonspecific lipid transfer proteins (nsLTPs) were found in plants .Up to now, we do not know their functions in vivo, but many researches have testified that these proteins can bind and catalyze transfer of lipids in vitro, which include phospholipids, glycolipids and fatty acids. A large number of these proteins have been isolated and their primary and secondary structures have been identified. These results showed these proteins have a high degree of similarity; they are basic and cysteine-rich proteins with a signal peptide and a common pattern of eight cysteines that engaged in four disulphide bridges holding together fourαhelices and stabilizing the structural fold. A hydrophobic central cavity in which occupied by lipids is found between the four helices. However, it has been difficult to draw any conclusions about the in vivo activity of nsLTPs from their lipid binding properties because it is unknown which ligands, if any, are bound to nsLTPs in vivo. These proteins are ubiquitous in the plant kingdom where they form a multiple genetic family. The suggestion that they would act as intracellular transporters of lipids between organelles, so, many different functions have been proposed for plant nsLTPs. They have been suggested to be involved in different aspects of plant physiology and cell biology through their ability to bind and/or carry lipophilic compounds, including the formation of cutin by transporting the hydrophobic cutin monomers to the apoplast and the defence of plants against pathogens as antimicrobial agents and in flowering. Also nsLTPs have been suggested to be important in several types of plant stress response including responses to drought, and temperature changes and cold and environmental changes. However, in fact, nsLTPs are extracellular proteins, so there is yet no direct demonstration of an in vivo function. Despite their implication in these diverse aspects of plant biology, it is not clear which specific role nsLTPs play here, because in a given plant, several nsLTP genes can be found, and they are often specifically expressed in both time and tissue. Also individual nsLTP genes are induced under a variety of conditions. It is not clear whether the various nsLTPs have functional overlap. If this is the case, it may prove difficult to use genetic approaches in determining the function of nsLTPs.We have identified a nucleotide sequence from ESTs (Expressed Sequence Tags) acquired form a cDNA library of Thellungiella halophila treated with 200mM/NaCl by the large-scale partial sequencing of randomly selected cDNA clones. This sequence emerges fourteen times in 1000 ESTs library. These result indict that it is a middle affluently gene in cDNA library. The cDNA of 634 basepairs contains an open reading frame of 339 nucleotides encoding a novel nonspecific lipid transfer protein .The first 23 amino acids constitute the putative signal peptide, characteristic for targeting to the secretory pathway. We analyzed their sequence characterizations, genomic structures and transcription levels under many stresses. The predicted amino acid sequence of T. halophila is highly similar to that of the other plant nsLTPs. Analysis of mRNA levels by Northern blotting indicated that the expression level of nsLTP in T. halophila leaves was significantly increased after being treated with different concentration of NaCl, and different hours with 200mmol/L NaCl. The transcription level although increased significantly after the treatment with different concentrations of ABA ,PEG and treatment with different hours of hot ( 37℃) and cold ( 4℃). These result show that this gene is a stress response gene or have important role in the tolerance stresses .In order to testify the function of the gene, we have cloned the gene into the Agrobacterium tumefaciens binary vectors pROKⅡand pCAMBIA 3301. A. tumefaciens GV3101 (pMP90) derivatives carrying these plasmids were used in genetic transformation of plants by flower dripping method. Chlorophyll leaching assays and fresh weight loss experiments indicated that overexpression of the Th-nsLTP4 genes reduced cuticle permeability, probably because of changes in its total wax amount on the surface cuticle in Arabidopsis, however silent expression of the Th-nsLTP4in T .halophila increase the permeability of cuticle by analysis Chlorophyll leaching. Overexpression Th-nsLTP4in Arabidopsis displayed significant drought and salt tolerance, probably related to the reduced epidermal permeability. In transgenic lines the fresh and dry weight increased and had more water content, Compared with the wild type.Transgenic lines displayed PEG and mannitol tolerance suggested overexpression of the Th-nsLTP4in Arabidopsis increase plant tolerance. The total wax amount on transgenic lines leaves and stems were increased and showed proportional increase in the alkanes, secondary alcohols and ketones. The total wax amount on transgenic T .halophila of silent expression of the Th-nsLTP4 leaves and stems was reduced and showed proportional deficiencies in the aldehydes, alkanes, secondary alcohols, and ketones . Besides altered cuticle wax amount, the transgenic lines had increased cuticle permeability.Compared with the wild type, Overexpression and silent expression of the Th-nsLTP4 all inflected the total wax load and epidermal permeability in Arabidopsis and T .halophila, respectively. Lower epidermal permeability most important functions is limiting transpiration to conserve water, a mechanism especially important for plant survival in water-limited environments such as those found in dehydration and salt stress. Overexpression Th-nsLTP4,a transfer lipid protein can increase total cuticular wax levels may because it can increase transfer speed of wax composition and feedback stimulate the synthesis of wax. Furthermore, as a cell wall localized protein, a amount of Th-nsLTP4deposited in cell wall might limit transpiration to conserve water by reduced permeability of cell wall.The YRE/WAX2 of Arabidopsis encodes an aldehyde-generating acyl-CoA enzyme in wax biosynthetic pathway. To further elucidate the function of WAX2 in cuticular waxes biosynthesis, we overexpressed the WAX2 using the strong constitutive CaMV 35S promoter in Arabidopsis. Compared with the wild type, transgenic plants failed to promote greater wax deposition on stems and leaves, and also showed higher transpiration rate, as well as reduced fertility under low humidity. These results suggested that error spatial, temporal expression or abnormality increased the levels of WAX2 could induce cosuppression of the endogenous genes and shut down the whole wax biosynthetic pathway.
|
PDF Full Text Request