Stasolla Claudio, Yeung Edward C
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2.
Tree Physiol. 2006 Sep;26(9):1197-206. doi: 10.1093/treephys/26.9.1197.
Previously, we demonstrated that, in some cell lines, exogenous applications of ascorbic acid (ASC) enhance the conversion frequency of white spruce (Picea glauca (Moench) Voss) somatic embryos, by stimulating mitotic activity in the apical meristems. To examine this event in more detail, we investigated the effects of ASC on de novo, salvage and degradation pathways of pyrimidine metabolism by following the metabolic fate of (14)C-labeled orotic acid, thymidine, uridine and uracil in shoot and root poles of germinating embryos, after altering the cellular ASC content of the embryos. Alterations in endogenous ASC content did not affect the utilization of either orotic acid or uracil, but affected the metabolism of thymidine and uridine. Specifically, a lowering of endogenous ASC content by applications of lycorine (L), an inhibitor of the last enzyme of the ASC de novo biosynthetic pathway, resulted in a lower embryo conversion frequency, as well as a reduced percentage of thymidine and uridine incorporated into nucleotides and nucleic acids. The reduction in thymidine and uridine anabolism was mainly ascribed to the decreased activities of thymidine kinase (TRK) and uridine kinase (URK), the respective salvage enzymes of thymidine and uridine, measured in L-treated embryos. These effects were solely a result of a decrease in endogenous ASC content because applications of ascorbic acid plus lycorine (ASC + L) increased embryo conversion frequency, thymidine and uridine salvage activities, and TRK and URK activities to near control values. Inclusion of exogenous ASC in the germination medium did not affect the percentage of embryos able to convert to viable plantlets, although it increased thymidine and uridine utilization for nucleic acid synthesis in the shoot and root poles of the embryos. Taken together, these findings confirm that cellular ASC plays a key role in the reactivation of the apical meristems of germinating white spruce somatic embryos.
此前，我们证明，在某些细胞系中，外源施加抗坏血酸（ASC）可通过刺激顶端分生组织中的有丝分裂活性，提高白云杉（Picea glauca (Moench) Voss）体细胞胚的转化频率。为了更详细地研究这一现象，我们通过追踪发芽胚的茎尖和根尖中（14）C标记的乳清酸、胸苷、尿苷和尿嘧啶的代谢命运，研究了ASC对嘧啶代谢的从头合成、补救和降解途径的影响，此前已改变了胚的细胞内ASC含量。内源性ASC含量的改变不影响乳清酸或尿嘧啶的利用，但影响胸苷和尿苷的代谢。具体而言，通过施加石蒜碱（L）降低内源性ASC含量，石蒜碱是ASC从头生物合成途径中最后一种酶的抑制剂，导致胚转化频率降低，以及掺入核苷酸和核酸中的胸苷和尿苷百分比降低。胸苷和尿苷合成代谢的降低主要归因于胸苷激酶（TRK）和尿苷激酶（URK）活性的降低，这两种酶分别是胸苷和尿苷的补救酶，在L处理的胚中进行了测定。这些影响完全是内源性ASC含量降低的结果，因为施加抗坏血酸加石蒜碱（ASC + L）可使胚转化频率、胸苷和尿苷补救活性以及TRK和URK活性增加至接近对照值。在发芽培养基中加入外源ASC并不影响能够转化为有活力小植株的胚的百分比，尽管它增加了胚的茎尖和根尖中用于核酸合成的胸苷和尿苷的利用率。综上所述，这些发现证实细胞内ASC在发芽的白云杉体细胞胚顶端分生组织的重新激活中起关键作用。
