Ultrastructural and Enzymic Studies of Cell Membranes from Ice-encased and Noniced Winter Wheat Seedlings

A marked increase in the amount of cisternal-like cytoplasmic membranes was observed after ice encasement of winter wheat (Triticum aestivum L.) seedlings. Linear sucrose gradients were employed to separate the various membrane components of the microsomal membrane fraction. NADH- and NADPH-cytochrome c reductase, two specific enzyme markers for plant endoplasmic reticulum (ER) were used to locate the ER in the linear gradients. The identity of the ER fraction was confirmed by determining the effect of EDTA and Mg²⁺ in the preparative media on the distribution of NADH- and NADPH-cytochrome c reductase activity within the gradient. In the presence of EDTA which dissociates ribosomes from ER, peaks of activity for the two enzymes were observed at a density corresponding to that for “smooth” ER. When the media also contained an appropriate concentration of Mg²⁺ to maintain the attachment of ribosomes to the ER, the peaks of activity for the enzymes shifted to a density corresponding to that for “rough” ER. NADH-cytochrome c reductase activity was similar for 24 C-grown and 2 C-grown iced seedlings, but significantly lower for 2 C noniced seedlings. No preferential increase in uptake of radioactive leucine or choline in the ER was observed during ice encasement. The accumulation of electron microscopically visible membrane arrays was not inhibited by the presence of protein synthesis inhibitors at concentrations which severely inhibited incorporation of 1-¹⁴C]leucine into membrane protein, but did not affect survival and growth of the seedlings. These observations indicate that the apparent proliferation of ER during ice encasement does not result from net membrane synthesis, but rather from reorganization of existing membrane elements within the cell.