Houde M, Daniel C, Lachapelle M, Allard F, Laliberté S, Sarhan F
Département des Sciences biologiques, Université du Québec à Montréal, Canada.
Plant J. 1995 Oct;8(4):583-93. doi: 10.1046/j.1365-313x.1995.8040583.x.
A protein family associated with the development of freezing tolerance in wheat has been identified. This protein family is Gramineae-specific and coordinately regulated by low temperature. Antibodies directed against the 50 kDa (WCS120) protein recognize at least 5 members of this family. Using these antibodies, the cellular content and location of this protein family was determined in cold-acclimated wheat seedlings. Western analyses of subcellular fractions indicated the presence of all members of the family in the cytosolic and purified nuclear fractions. These proteins accumulated to 0.9% of soluble proteins after 21 days of cold acclimation in winter wheat. This represents a cellular concentration of 1.34 microM. Immunohistochemical localization showed that these proteins are highly expressed in the vascular transition zone. No detectable expression was found in mature xylem, in the shoot apical meristem or lateral root primordia. This differential tissue expression suggests that the sensitive cells near the regions where water tends to freeze first require a higher amount of these proteins. This observation is consistent with the fact that regrowth after freezing stress is highly dependent on the viability of this region of the crown. Electron microscopy analysis using immunogold labelling showed that these proteins are present in the cytoplasm and in the nucleoplasm. They are not found in cell walls or other organelles. In vitro cryoprotective assays indicated that the WCS120 protein (PD50 of 10 micrograms ml-1 or 0.2 microM) are as effective as BSA and sucrose (at 250 mM) against freezing denaturation of lactate dehydrogenase. These results suggest that this protein family may be involved in a general mechanism of protection in the soluble fraction of the cell. Their presence in the nucleoplasm may also suggest a possible protective function of the transcriptional machinery. The high hydrophilicity, the abundance and stability of these proteins to boiling suggest that they may provide a particular micro-environment needed for cell survival in the sensitive vascular transition zone during freezing stress.
已鉴定出一个与小麦抗冻性发育相关的蛋白质家族。该蛋白质家族是禾本科特有的，受低温协同调控。针对50 kDa（WCS120）蛋白质的抗体识别该家族的至少5个成员。利用这些抗体，在冷驯化的小麦幼苗中确定了该蛋白质家族的细胞含量和定位。亚细胞组分的蛋白质免疫印迹分析表明，该家族的所有成员都存在于胞质和纯化的核组分中。在冬小麦冷驯化21天后，这些蛋白质积累至可溶性蛋白质的0.9%。这代表细胞浓度为1.34微摩尔。免疫组织化学定位表明，这些蛋白质在维管束过渡区高度表达。在成熟木质部、茎尖分生组织或侧根原基中未检测到表达。这种不同的组织表达表明，靠近水最先结冰区域的敏感细胞需要更高量的这些蛋白质。这一观察结果与冻害后再生高度依赖于冠部该区域活力的事实一致。使用免疫金标记的电子显微镜分析表明，这些蛋白质存在于细胞质和核质中。在细胞壁或其他细胞器中未发现它们。体外冷冻保护试验表明，WCS120蛋白质（半数保护剂量为10微克/毫升或0.2微摩尔）在防止乳酸脱氢酶冷冻变性方面与牛血清白蛋白和蔗糖（250毫摩尔）一样有效。这些结果表明，该蛋白质家族可能参与细胞可溶性部分的一般保护机制。它们在核质中的存在也可能表明转录机制具有潜在的保护功能。这些蛋白质的高亲水性、丰富性和对煮沸的稳定性表明，它们可能为冷冻胁迫期间敏感维管束过渡区的细胞存活提供所需的特定微环境。
