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Recently, five novel fluorescent proteins have been isolated from non-bioluminescent species of reef-coral organisms and have been made available through ClonTech. They are AmCyan, AsRed, DsRed, ZsGreen and ZsYellow. These proteins are valuable as reporters for transformation because they do not require a substrate or external co-factor to emit fluorescence and can be tested in vivo without destruction of the tissue under study. We have evaluated them in a large range of plants, both monocots and dicots, and our results indicate that they are valuable reporting tools for transformation in a wide variety of crops. We report here their successful expression in wheat, maize, barley, rice, banana, onion, soybean, cotton, tobacco, potato and tomato. Transient expression could be observed as early as 24 h after DNA delivery in some cases, allowing for very clear visualization of individually transformed cells. Stable transgenic events were generated, using mannose, kanamycin or hygromycin selection. Transgenic plants were phenotypically normal, showing a wide range of fluorescence levels, and were fertile. Expression of AmCyan, ZsGreen and AsRed was visible in maize T1 seeds, allowing visual segregation to more than 99% accuracy. The excitation and emission wavelengths of some of these proteins are significantly different; the difference is enough for the simultaneous visualization of cells transformed with more than one of the fluorescent proteins. These proteins will become useful tools for transformation optimization and other studies. The wide variety of plants successfully tested demonstrates that these proteins will potentially find broad use in plant biology.

Wild-type and mutated forms of the wheat (Triticum aestivum L.) storage protein gamma-gliadin were expressed in transgenic tobacco (Nicotiana tabacum L. cv. NVS) under the control of the 35S cauliflower mosaic virus (CaMV) promoter in order to determine what, if any, endogenous targeting signals are present in the wild-type gamma-gliadin protein. The mutant forms of the protein were modified by the addition of a KDEL or HDEL C-terminal endoplasmic reticulum-retention signal, or the addition of a C-terminal propeptide from barley lectin which has been shown to be necessary and sufficient for targeting to the vacuole. Only modified forms of the protein accumulated in leaves of transgenic tobacco, although the transcript levels were similar for all the constructs. Pulse-chase analysis indicated that whereas the wild-type gamma-gliadin was rapidly turned over in tobacco leaves, KDEL and HDEL forms were highly stable. The vacuolar-signal mutant protein accumulated in tobacco leaves, but migrated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis with a lower mobility than wild-type gamma-gliadin, due in part to glycosylation of the C-terminal propeptide. The vacuolar-signal mutant protein was turned over slowly in tobacco, perhaps indicating a poor level of transport competence. When pulse-chase analysis was carried out on protoplasts isolated from tobacco plants expressing wild-type gamma-gliadin, but in the presence of Brefeldin A, gamma-gliadin was seen to accumulate. Taken together, these results indicate that gamma-gliadin is targeted to the vacuole in transgenic tobacco plants and does not contain any structural determinants which confer retention in the endoplasmic reticulum.

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