chapter  11
12 Pages

USE OF XENOPUS OOCYTES TO MONITOR PLANT GENE EXPRESSION

While the Xenopus oocyte microinjection system has been used primarily in the study of animal gene expression. recently it has gained popularity in the analysis of plant gene expression. A summary of plant mRNAs isolated from various tissues, plant viruses as well as synthetic mRNAs made from cloned plant cDNAs. which have been successfully translated in Xenopus oocytes is given in Table I. For example, oocytes injected with barley mRNAs can synthesize and secrete catalytically active barley a-amylase. 16 Also, RNA isolated from elicitor-treated tomato cells has directed the expression of an ethylene-forming enzyme (EFE) when injected into oocytes, as judged by their ability to convert 1-aminocyclopropane-1carboxylic acid into ethylene. 15 In fact, EFE expressed in oocytes was indistinguishable from tomato cell EFE with respect to its saturation kinetics, iron dependency, sensitivity to inhibitors, stereospecificity, and inducibility by fungal elicitors. Microinjection of plant mRNA synthesized in vitro has been employed to evaluate the effect of mutagenized mRNA on translation and post-translational modifications. When artificially mutagenized maize protein mRNA was translated in Xenopus oocytes, it was demonstrated that the addition of tryptophan and lysine to zein. which normally lacks these amino acids. does not affect the translation, post-translational modifications, or stability of the mutant protein.~0 This finding suggested that the creation of high lysine com by genetic engineering was feasible. Recently, synthetic mRNAs made from eDNA clones for carrot and soybean cyclins (proteins which are required for mitosis or meiosis) were shown to cause Xenopus oocyte maturation following injection. 17

Thus, certain plant proteins may have comparable functions in animal cells. In addition to translating plant mRNA, the Xenopus oocyte system has been used to analyze

the expression of injected plant DNA (Table 1). The regulatory regions for the cauliflower mosaic virus 35S and for nopaline synthase were each fused to the bacterial reporter gene, chloramphenicol acetyl transferase (CAT) and microinjected into the germinal vesicles or nuclei of Xenopus oocytesY These DNA constructs actively supported the synthesis of CAT, demonstrating that promoter regions from plant genes can support transcription in Xenopus oocytes. Also, a comprehensive study of barley a-amylase gene expression was carried out

Al RNA isolated from plant tissue• Castor bean lectin Maize storage proteins

Common bean storage protein Bean phytohemaglutinin Barley seed low mol. w!. proteins Field bean storage globulins French bean storage globulins Pea bean storage globulins Tomato ethylene-forming enzyme Barley a-amylase

B) Synthetic plant mRNAb Tomato ethylene-forming enzyme Carrol cydin Soybean cyclin 19 kDa maize zein protein Barley a-amylase

C) Plant viral RNA Tobacco mosaic virus Cow pea mosaic virus Alfalfa mosaic virus Brome mosaic virus

Dl Plant genomic DNA constructs Barley a-amylase Nopaline synthase' Cauliflower mosaic virus 35S'.