He Xu, Kermode Allison R
Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, B.C., Canada V5A 1S6.
Plant Mol Biol. 2003 Jul;52(4):729-44. doi: 10.1023/a:1025008117046.
During post-germinative seedling growth, the major storage organ of the white spruce (Picea glauca) seed, the megagametophyte, undergoes programmed cell death (PCD). Protease activities in megagametophyte cells that arise post-germinatively were investigated. The accumulation of protease activities can be divided into two phases: the first phase correlated with degradation of storage proteins while the second phase was temporally associated with cell death, although some of the early proteases were also active during the later phase. Proteases induced during PCD were mainly serine and cysteine proteases. One of the PCD-associated cysteine proteases had homology to Cys-EP, a PCD-related cysteine protease of the castor bean endosperm. Transcripts encoding a Cys-EP-related protein were not present in megagametophytes when seeds were imbibed, nor were they present during germination and early post-germinative growth (radicle length ca. 2-5 mm). At a later post-germinative stage (i.e when the seed's radicle was ca. 15 mm), the Cys-EP-related transcripts (ca. 1.3 kb) became abundant and, at this time, the 48 kDa proform of the enzyme first appeared. The mature form of the Cys-EP (ca. 38 kDa) was predominant at a very late stage of post-germinative growth. Immunocytochemistry showed that the Cys-EP-related protein was localized to spherical organelles (ca. 2 microm) that may be equivalent to the 'ricinosomes' of castor bean endosperm cells. Caspase-like protease (CLP) activities were first detected 3 days after germination with the caspase-specific substrate Ac-DEVD-AMC; maximum activities occurred when the seed's radicle was ca. 20-25 mm. When germinated seeds were treated with a caspase-3 inhibitor, both the peak of CLP activities and the death of megagametophyte cells were delayed. We propose that the Cys-EP-related protein and CLP activity are involved in PCD of white spruce megagametophyte cells.
在发芽后的幼苗生长过程中，白云杉（Picea glauca）种子的主要储存器官——雌配子体，会经历程序性细胞死亡（PCD）。对发芽后雌配子体细胞中的蛋白酶活性进行了研究。蛋白酶活性的积累可分为两个阶段：第一阶段与储存蛋白的降解相关，而第二阶段在时间上与细胞死亡相关，尽管一些早期蛋白酶在后期也有活性。PCD过程中诱导产生的蛋白酶主要是丝氨酸蛋白酶和半胱氨酸蛋白酶。其中一种与PCD相关的半胱氨酸蛋白酶与蓖麻籽胚乳中与PCD相关的半胱氨酸蛋白酶Cys-EP具有同源性。种子吸胀时，雌配子体中不存在编码Cys-EP相关蛋白的转录本，在萌发和发芽后的早期生长阶段（胚根长度约2-5毫米）也不存在。在发芽后的后期阶段（即种子胚根约15毫米时），Cys-EP相关转录本（约1.3 kb）变得丰富，此时该酶的48 kDa前体形式首次出现。Cys-EP的成熟形式（约38 kDa）在发芽后生长的非常后期占主导地位。免疫细胞化学显示，Cys-EP相关蛋白定位于球形细胞器（约2微米），可能等同于蓖麻籽胚乳细胞的“蓖麻毒蛋白体”。发芽3天后，用半胱天冬酶特异性底物Ac-DEVD-AMC首次检测到类半胱天冬酶蛋白酶（CLP）活性；当种子胚根约20-25毫米时，活性达到最大值。用半胱天冬酶-3抑制剂处理发芽的种子时，CLP活性峰值和雌配子体细胞死亡均延迟。我们认为，Cys-EP相关蛋白和CLP活性参与了白云杉雌配子体细胞的PCD。
