Chapman A, Blervacq A S, Vasseur J, Hilbert J L
Laboratoire de Physiologie Cellulaire et Morphogenèse Végétales, USTL/INRA, Université des Sciences et Technologies de Lille, Villeneuve d'Ascq, France.
Planta. 2000 Aug;211(3):305-14. doi: 10.1007/s004250000299.
Direct somatic embryogenesis was induced in root tissues of the Cichorium hybrid '474' (C. intybus L. var. sativum x C. endivia L. var. latifolia). Addition of beta-D-glucosyl Yariv reagent (betaGlcY), a synthetic phenylglycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in a concentration-dependent manner with complete inhibition of induction occurring at 250 microM betaGlcY. The AGP-unreactive alpha-D-galactosyl Yariv reagent had no biological activity in this system. Upon transfer of 250 microM betaGlcY-treated roots to control conditions, somatic embryogenesis was recovered with a time course similar to that of control roots. The betaGlcY penetrated roots and bound abundantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (JIM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell walls peripheral cells of the globular embryo. A spatio-temporal expression of AGPs appeared to be associated with differentiation events in the somatic embryo during the transition from the globular stage to the torpedo stage. To verify betaGlcY specificity, molecules that bound betaGlcY were extracted from treated conditioned medium and identified as AGPs by using the same monoclonal antibodies. In addition, AGPs were found to be abundantly present in the medium during embryogenic culture. All of these results establish the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed.
在菊苣杂交种“474”(C. intybus L. var. sativum×C. endivia L. var. latifolia)的根组织中诱导出了直接体细胞胚胎发生。向培养基中添加β-D-葡萄糖基Yariv试剂(βGlcY),一种特异性结合阿拉伯半乳聚糖蛋白(AGPs)的合成苯基糖苷,以浓度依赖的方式阻断了体细胞胚胎发生,在250μMβGlcY时完全抑制了诱导。AGP无反应性的α-D-半乳糖基Yariv试剂在该系统中没有生物学活性。将经250μMβGlcY处理的根转移到对照条件下后,体细胞胚胎发生得以恢复,其时间进程与对照根相似。βGlcY穿透根并大量结合到发育中的体细胞胚、根表皮和中柱上。使用单克隆抗体(JIM13、JIM16和LM2)进行的免疫荧光和免疫金标记显示,AGPs定位于球形胚外周细胞的外细胞壁中。AGPs的时空表达似乎与体细胞胚从球形阶段向鱼雷阶段转变过程中的分化事件相关。为了验证βGlcY的特异性,从处理后的条件培养基中提取了与βGlcY结合的分子,并使用相同的单克隆抗体将其鉴定为AGPs。此外,在胚胎发生培养过程中发现培养基中大量存在AGPs。所有这些结果都证实了AGPs在胚胎发育中的作用,并讨论了它们在体细胞胚胎发生中的假定作用。