The product of the rice myb7 unspliced mRNA dimerizes with the maize leucine zipper Opaque2 and stimulates its activity in a transient expression assay.

myb7 mRNA is present in rice in spliced and unspliced forms, splicing being enhanced by anoxia. The protein (Mybleu) encoded by the unspliced mRNA is composed of an incomplete Myb domain followed by a leucine zipper; however, it lacks canonical sequences for DNA binding, transcriptional activation, and nuclear localization. We show here that in transiently transformed tobacco protoplasts, Mybleu is able to enhance the transcriptional activity of the maize leucine zipper Opaque2 on its target b32 promoter. The Mybleu transactivation effect is strictly dependent on the presence of Opaque2 and is driven by Mybleu-Opaque2 heterodimers. Mybleu is located in the nucleus, both in rice and in transformed tobacco protoplasts. In rice, the protein is expressed in regions corresponding to undifferentiated cells of roots and coleoptiles. Therefore, myb7 mRNA encodes, depending on its splicing, two transcription factors belonging to separate classes. One of them, Mybleu, has novel structural characteristics, suggesting the existence of new mechanisms acting in the activation of transcription.

Functional analysis of the regulatory region of a zein gene in transiently transformed protoplasts.

The transcription of zein genes in maize is tissue-specific and developmentally regulated. The 5' regulatory region of many zein genes contains two promoters, P1 and P2, lying approximately 1000 bases apart. The promoter/enhancer activity of various fragments of the two promoter regions of the zein gene E19 have been analysed by means of transient expression experiments. The results indicate that the various regions differentially affect the expression of the GUS reporter gene activity in protoplasts from tobacco leaves, maize immature endosperms and in vitro endosperm cell cultures. In tobacco protoplasts only the proximal promoter region, P2, activates GUS expression, while in endosperm culture cells only the distant promoter, P1, gives significant activity. The P1 region, both in direct and opposite orientation, stimulates a low level of GUS expression in protoplasts from immature endosperms.

Stable endosperm cell suspension cultures from wild-type and opaque-2 maize.

Stable cell suspension cultures have been established from immature endosperms of A69Y wild-type and opaque-2 maize (Zea mays L.). Cultured cells are capable of storage protein (zein) synthesis and accumulation throughout the growth period. Electrophoretic patterns of zeins show, for opaque-2 cells, the preferential inhibition of the accumulation of 22 kDa peptides typical of the mutation. Viable protoplasts, able to regenerate cell walls, as well as to divide and to express foreign DNA in transient expression experiments, can be obtained with high yields from cultures of both genotypes.

Expression of zein in long term cultures of wildtype and opaque-2 maize endosperms.

It has been reported that long term cell cultures from maize endosperms are not completely de-differentiated, maintaining some tissue-specific synthesis. We analyzed the expression of zein (the major storage protein of maize seeds) in cultures derived from wildtype and opaque-2 maize endosperms. In wildtype cultures, our data indicate a severe restriction in zein accumulation, resulting both from reduction of transcription and from post-transcriptional events. No detectable zein proteins and trace amounts of transcripts were found in opaque-2 cultures, which do not therefore exhibit a distinctive opaque-2 phenotype.

Anthocyanin and proteins as biochemical markers in maize endosperm cultures.

Endosperm maize cultures derived from a strain homozygous for all genes required for anthocyanin synthesis develop an intense pigmentation. Pigmenting ability is generally maintained in successive subcultures, altough colourless areas are frequently observed in pigmented cultures. The isolated colourless cell clusters show a growth rate higher than the coloured ones. These calli nevertheless do not lose the ability to synthesize anthocyanins, and in successive subcultures turn red again.The different growth rates associated with the ability of cells to accumulate pigments suggest the existence of different physiological states of the culture. To investigate this possibility we analyzed the polypeptide patterns of coloured and colourless cultures. SDS gel electrophoresis has demonstrated differences in soluble protein fractions, among which a 26 kD peptide, characteristic of pigmented tissues, has been evidenced. Zein, the major storage protein of maize endosperm is present, although at very low levels, both in pigmented and in unpigmented cultures, confirming that its synthesis occurs continuously in vitro.

Subcellular distribution and functional properties of different forms of elongation fractor EF1 from wheat embryos.

Elongation factor EF1 was found in a low salt homogenate of wheat embryos, either in the 100 000 X g supernatant or in the ribosome pellet. The ribosome-linked EF1 (EF1R), deteched by high salt washing, was purified to electrophoretical homogenetiy and its molecular and functional properties compared to those of a purified high molecular weight species of EF1 obtained from cytoplasm (EF1H). The two forms are associations of different polypeptides having in common only the polypeptide which can form the ternary complex with aminoacyl-tRNA and GTP. Whereas EF1R is able to fulfill all the EF1 functions, EF1H, incubated with ribosomes completely deprived of elongation factors, can catalyze the aminoacyl-tRNA binding to ribosomes, but, in the presence of EF2, forms only a very small amount of poly(Phe).