Role of the leader sequence in tobacco pectin methylesterase secretion.

We report that unprocessed tobacco pectin methylesterase (PME) contains N-terminal pro-sequence including the transmembrane (TM) domain and spacer segment preceding the mature PME. The mature portion of PME was replaced by green fluorescent protein (GFP) gene and various deletion mutants of pro-sequence fused to GFP were cloned into binary vectors and agroinjected in Nicotiana benthamiana leaves. The PME pro-sequence delivered GFP to the cell wall (CW). We showed that a transient binding of PME TM domain to endoplasmic reticulum membranes occurs upon its transport to CW. The CW targeting was abolished by various deletions in the TM domain, i.e., anchor domain was essential for secretion of GFP to CW. By contrast, even entire deletion of the spacer segment had no influence on GFP targeting.

New CMS-associated phenotypes in cybrids Nicotiana tabacum L. (+Hyoscyamus niger L.).

Morphological characteristics were studied in cytoplasmic male sterile (CMS) cybrids possessing the tobacco nuclear genome, Hyoscyamus niger plastome and recombinant mitochondria. After backcrosses with tobacco, new flower modifications were found, including: conversions of stamens into branched filamentous structures; alterations in the shape of petals and the corolla limb; and high degrees of reduction in most flower organs. Vegetative alterations (leaf elongation and stem branching) occurred in some cybrids. Results confirmed that a protoplast fusion-based alloplasmic cytoplasm transfer, followed by conventional backcrosses, is a useful tool for generating alternative CMS sources with novel nucleo-cytoplasmic compositions. These alterations in the genetic status were accompanied by modified floral and vegetative phenotypes.