mRNAs newly synthesized by tobacco mesophyll protoplasts are wound-inducible

Abstract

We have used 2-dimensional (2D) non-equilibrium pH gradient gel electrophoresis (NEPHGE) of in vitro synthesized proteins and northern hybridization with labelled cDNAs coding for three pathogenesis related (P.R.) proteins, to analyze the shift in mRNA content induced by the isolation and culture of tobacco mesophyll protoplasts. The in vitro protein pattern of mRNAs from freshly isolated protoplasts is characterized by the absence of most leaf spots and the appearance of 19 new spots. After 6 hours of culture, the mRNAs coding for the P.R. proteins become detectable and after 12 hours the protoplasts contain an mRNA population almost typical of callus cells. The different steps involved in the isolation and culture of protoplasts were analysed. Cutting off the leaf and sterilization do not change the mRNA set. In contrast, the mechanical injury applied to the leaf in order to facilitate the penetration of the enzymatic mixture induces a modification of the mRNA content identical to that resulting from protoplast isolation. Wounding is the essential event inducing dedifferentiation. Varying the culture medium and conditions leads to only limited modifications of the mRNA pattern. These results are discussed on the basis of present knowledge of the reaction of the plant to wounding and we suggest that wound healing callus and in vitro callus correspond to the same differentiation state.

Triticum aestivum puroindolines, two basic cystine-rich seed proteins: cDNA sequence analysis and developmental gene expression

“…Total R N A was extracted from seeds (2.5 g) as described by Grosset et al [20]. Before electrophoresis R N A samples were vacuum-dried, dissolved in 0.15 M M O P S and 3 mM EDTA, then formaldehyde and formamide were added to a final concentration of 2.2 M and 50~o (v/v), respectively [15].…”

Laser-Capture Microdissection, a Tool for the Global Analysis of Gene Expression in Specific Plant Cell Types: Identification of Genes Expressed Differentially in Epidermal Cells or Vascular Tissues of Maize[W]

“…In general, however, the preparation of protoplasts from intact plant tissues is time consuming. Of greater concern is the fact that gene expression patterns change during protoplast preparation (Grosset et al, 1990). Thus, this method is unlikely to be useful for monitoring gene expression in specialized cell types.…”

Identification of proteins related to microspore embryogenesis responsiveness in anther cultures of winter triticale (×Triticosecale Wittm.)

“…The role of such a protein is elusive, although its accumulation could be caused by mechanical damage during the anthers isolation. Wounding itself could be a significant signal for dedifferentiation, causing the change in mRNA content for example in tobacco mesophyll protoplast cultures (Grosset et al 1990).…”