Efficiency of transient transformation in tobacco protoplasts is independent of plasmid amount.

We describe an optimized protocol for the transient transformation of tobacco protoplasts mediated by polyethylene-glycol (PEG). As expected, the quantitative beta-glucuronidase (Gus) activity driven by pCaMVGus was dependent on the amount of plasmid used. Nevertheless, we demonstrate by an immunodetection method that transformation efficiency did not depend on the amount of plasmid used but on the limitation imposed by cell competence. In fact, we obtained the same percentage of transformed cells (about 60%) using a wide range of plasmid concentrations (0.1-10 microg per test). Finally, we show that, when we used two plasmid types in a mixture at a concentration ranging from 0.1 to 10 microg for each, all transformed cells expressed proteins encoded by both plasmids. Transient expression and co-transformation experiments are routinely used methods and, probably, the major results from this work were assumed by many researchers in this field, but our data experimentally support this assumption.

Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase.

Increased ethylene evolution accompanies seed germination of many species including Pisum sativum L., but only a little is known about the regulation of the ethylene biosynthetic pathway in different seed tissues. Biosynthesis of the direct ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), the expression of ACC oxidase (ACO), and ethylene production were investigated in the cotyledons and embryonic axis of germinating pea seeds. An early onset and sequential induction of ACC biosynthesis, accumulation of Ps-ACO1 mRNA and of ACO activity, and ethylene production were localized almost exclusively in the embryonic axis. Maximal levels of ACC, Ps-ACO1 mRNA, ACO enzyme activity and ethylene evolution were found when radicle emergence was just complete. Treatment of germinating seeds with ethylene alone or in combination with the inhibitor of ethylene action 2,5-norbornadiene showed that endogenous ethylene regulates its own biosynthesis through a positive feedback loop that enhances ACO expression. Accumulation of Ps-ACO1 mRNA and of ACO enzyme activity in the embryonic axis during the late phase of germination required ethylene, whereas Ps-ACS1 mRNA levels and overall ACC contents were not induced by ethylene treatment. Ethylene did not induce ACO in the embryonic axis during the early phase of germination. Ethylene-independent signalling pathways regulate the spatial and temporal pattern of ethylene biosynthesis, whereas the ethylene signalling pathway regulates high-level ACO expression in the embryonic axis, and thereby enhances ethylene evolution during seed germination.

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.

Maturation and translation mechanisms involved in the expression of a myb gene of rice.

We have isolated two overlapping cDNAs coding for a MYB-related protein expressed in aerobic and anaerobic rice (Oryza sativa) roots and coleoptiles. Analysis of their sequences reveals some peculiar features, suggesting the presence of post-transcriptional regulation events: an upstream ORF, two unspliced introns and a putative leucine zipper in the ORF coded by the unspliced RNA. Transient expression in protoplasts indicates that the upstream ORF inhibits expression of a downstream coding sequence. Finally, we demonstrated that anoxia, in roots, increases the ratio between the spliced and the unspliced mRNA and affects the expression of other myb-related genes.

Expression of the Enzymes of Nitrate Reduction during the Anaerobic Germination of Rice.

During the anaerobic germination of rice (Oryza sativa L.), nitrate is translocated from the caryopsis and assimilated into the coleoptile (R. Reggiani, M. Mattana, N. Aurisano, A. Bertani [1993] Plant Cell Physiol 34: 379-383). Using antibodies against nitrate and nitrite reductases, proteins with the expected molecular mass were recognized by western blot analysis in extracts from 8-d-old rice coleoptiles. Both enzymes are de novo synthesized in 6- to 8-d-old seedlings, as shown by immunoprecipitation of radiolabeled proteins from young plants grown in the presence of [35S]methio-nine. The anaerobic synthesis of both enzymes was enhanced by the addition of 5 mM KNO3. The effect of exogenous nitrate on the expression of the corresponding genes in anaerobic rice coleoptiles was revealed by the analysis of their transcripts. The importance of the expression of these enzymes during the anaerobic development of rice seedlings is discussed.

Rice membranes contain a calcium-dependent protein kinase activity with biochemical features of animal protein kinase C.

The presence of calcium-dependent protein kinase activities in rice was investigated. Membrane preparations could phosphorylate the MARCKS peptide, a highly specific substrate for animal protein kinase C (PKC). Phosphorylation, strictly dependent on calcium, was specifically antagonized by a peptide whose amino acid sequence corresponds to the inhibitory, pseudosubstrate domain of mammalian PKC. Similar results have been obtained with rice soluble fractions. Addition of inhibitors of mammalian PKC (staurosporine and calphostin C) also inhibited phosphorylation of specific peptide substrates. Western blot analysis with anti-PKC antibodies identified three major bands (90, 87 and 54 kD) in rice membrane-associated proteins.

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.

Auxin and growth regulation of rice coleoptile segments : molecular analysis.

Auxin-stimulated elongation of apical segments of rice (Oryza sativa L. cv Arborio) coleoptiles occurring in the first 4 hours of treatment has been studied. Cell extension promoted in the first 2 hours by 10 micromolar indole-3-acetic acid (IAA) is specifically auxin-dependent, whereas after 4 hours, elongation also depends on endogenous production of ethylene. Similar to other systems, rice coleoptile cell elongation stimulated by auxin requires continuous synthesis of RNA and protein. Two-dimensional gel analysis of the in vitro translation products obtained from polyadenylated RNAs extracted from treated and untreated segments after 1 or 4 hours from the initial addition of IAA shows few transcriptional differences. At 60 minutes of treatment, the level of three mRNAs coding for proteins of 22.5, 25, and 33 kilodaltons was moderately enhanced while the disappearance of a 38 kilodalton translation product was observed. Additional repression of another mRNA coding for a 28 kilodalton product begins to show by this time, but becomes more evident after 4 hours treatment. At 4 hours, four IAA-specific mRNA enhancements coding for proteins with molecular masses ranging between 35 to 40 kilodaltons were also observed. We discuss these data in relation to the possible involvement of IAA-mediated transcriptional regulation in growth promotion of rice coleoptiles and, more widely, in control of cell elongation.

Pattern of expression of meristem-specific cDNA clones of barley (Hordeum vulgare L.).

Deoxyribonucleic-acid sequences expressed at high levels in meristematic tissues of barley (Hordeum vulgare L.) have been cloned by differential hybridization. Five out of the seven cDNA clones studied showed homologies to histone genes H2a (two clones), H2b, H3 and H4. Their patterns of expression, as studied by RNA and in-situ hybridization, were typical for genes transcribed during cell division. A sixth cDNA clone, Sab2, had a 65.7% identity (on a protein basis) to L2-like ribosomal proteins of Escherichia coli and other lower prokaryotes. In a domain of 50 amino acids, the seventh clone, Sab35, showed 69.0% sequence identity to the ribosomal protein L21 of Rattus norvegicus. The Sab35 mRNA contained in its 5'-untranslated leader sequence small open reading frames, a feature pointing to a possible translational control. The Sab35 in-situ hybridization pattern was to a certain degree different from that of the histone-like clone Sab11: it detected transcripts not only in tissues that are associated with vegetative and reproductive apices but also in sub-apical regions. The visualization in situ of transcripts coded by Sab11, 35 and 44 is discussed as a possible technique for studying differential gene expression in barley meristematic tissues.

The maize zein gene zE19 contains two distinct promoters which are independently activated in endosperm and anthers of transgenic Petunia plants.

The activity, tissue specificity and temporal expression of the tandem promoter region preceding a maize zein gene (zE19, encoding a 19 kDa zein protein) were tested in transgenic Petunia plants. To simplify the analysis, the tandem promoter as well as each of the two separate promoter regions were fused to the beta-glucuronidase (GUS) reporter gene. All of the three constructs directed the synthesis of GUS in the endosperm of transformed seeds indicating that both separate promoters are independently activated and show the same tissue and cell type specificity observed for zein genes in maize. The kinetics of accumulation and the localization of GUS activity are not coordinated with those of Petunia endogenous seed storage proteins during the development of transformed seeds. Unexpectedly, we detected high levels of GUS activity in anthers of transformed Petunia plants for all three constructs. This appears to reflect the expression pattern of zein genes in maize, since we detect zein transcripts in anthers. Finally, we discuss the possible origin and function of the tandem promoter arrangement on the basis of these data.

Translational regulation of the expression of zein cloned in yeast under an inducible GAL promoter.

Yeast cells transformed by a plasmid containing a zein sequence fused to an hybrid yeast promoter GAL1-10/CYC1 accumulate, during a batch growth in galactose minimal medium, large amounts of zein only during a growth-limited phase that precede the entering into the stationary phase. We found that zein is fairly stable in yeast cells and the increased accumulation of zein polypeptide depends mainly upon a marked increase of its rate of synthesis. The increase of the rate of heterologous protein synthesis is not dependent on variation in the plasmid copy number and it is not related to the relative level of zein mRNA, indicating the existence of a postranscriptional regulation that modulates the translatability of this messenger RNA in function of the growth conditions. A possible explanation of this modulation is discussed in terms of a codon bias effect that slow-down the translation of heterologous mRNAs during the exponential phase of growth.

Transcription and expression of zein sequences in yeast under natural plant or yeast promoters.

Maize genomic fragments containing the regulatory and coding regions of a zein gene for a low size class 23-kd protein have been inserted in an interspecific Escherichia coli-Saccharomyces cerevisiae expression vector in different constructions. The presence of the inducible GAL1-10 upstream activation site (UAS) allows us to regulate differentially by carbon sources the transcription of the zein gene both under the plant promoter and under the yeast CYC-1 promoter. We found that the zein promoter region is properly recognized at the correct transcription start, while different termination points occur during transcription. The yeast UAS was also shown to function as a typical eukaryotic enhancer regardless of its distance or orientation with respect to the plant promoter. Yeast cells transformed by a plasmid containing a zein sequence fused to a short piece of the CYC-1 gene produced a fused polypeptide, of expected mol. wt, in variable amount from 0.2 to 5% depending on the growth phase conditions.