Department of Botany, University of Georgia, 30602, Athens, GA, USA.
Planta. 1983 Dec;159(6):534-44. doi: 10.1007/BF00409143.
An enzyme-linked immunosorbent assay (ELISA), which uses both rabbit polyclonal and mouse monoclonal antibodies to phytochrome, has been adapted for quantitation of phytochrome in crude plant extracts. The assay has a detection limit of about 100 pg phytochrome (<1 fmol of monomer) and can be completed within 10 h. Nonspecific interference by crude plant extracts was detected and corrected for. Quantitation of phytochrome in crude extracts of etiolated oat (Avena sativa L.) seedlings by ELISA gave values that agreed well with those obtained by spectrophotometric assay. When etiolated oat seedlings were irradiated continuously for 24 h, the amount of phytochrome detected by ELISA and by spectrophotometric assay in crude extracts of these seedlings decreased by more than 1000-fold and about 100-fold, respectively. This discrepancy indicates that phytochrome in light-treated plants may be antigenically distinct from that found in fully etiolated plants. Both a decrease in the light and an increase in the dark of phytochrome content was observed in crude extracts of light-grown oat shoots, both green and Norflurazon-bleached, in response to a 12:12-h light-dark cycle. When these light-grown oat seedlings were kept in darkness for 48 h, phytochrome content detected by ELISA increased by 50-fold in crude extracts of green oat shoots, but only about 12-fold in extracts of herbicide-treated oat shoots. Phytochrome reaccumulation in green oat shoots was initially more rapid in the more mature cells of the primary leaf tip than near the basal part of the shoot. The inhibitory effect of Norflurazon on phytochrome accumulation was much more evident near the leaf tip than the shoot base. A 5-min red irradiation of oat seedlings at the end of a 48-h dark period resulted in a subsequent, massive decrease in phytochrome content in crude extracts from both green and Norflurazon-bleached oat shoots. These observations eliminate the possibility that substantial accumulation of chromophore-free phytochrome was being detected and indicate that Norflurazon has a substantial effect on phytochrome accumulation during a prolonged dark period.
酶联免疫吸附测定（ELISA），该方法使用兔多克隆和鼠单克隆抗体检测光敏色素，已经被用于定量测定粗植物提取物中的光敏色素。该测定法的检测限约为 100 pg 光敏色素（<1 fmol 单体），可在 10 小时内完成。检测到并纠正了粗植物提取物的非特异性干扰。通过 ELISA 定量测定黄化燕麦（Avena sativa L.）幼苗粗提物中的光敏色素，得到的值与分光光度法测定的值吻合较好。当黄化燕麦幼苗连续照射 24 小时时，ELISA 和分光光度法在这些幼苗粗提物中检测到的光敏色素含量分别下降了 1000 多倍和 100 倍以上。这种差异表明，光处理植物中的光敏色素可能与其在完全黄化植物中发现的光敏色素在抗原性上有所不同。在响应 12:12 小时的光暗循环时，在绿光和 Norflurazon 漂白的燕麦绿茎粗提物中都观察到光下和暗下光敏色素含量的减少。当这些绿苗燕麦幼苗在黑暗中放置 48 小时时，ELISA 在绿苗燕麦茎粗提物中检测到的光敏色素含量增加了 50 倍，但在除草剂处理的燕麦茎粗提物中仅增加了约 12 倍。在绿苗燕麦茎中，光敏色素的重新积累在初始时比在茎的基部更迅速地在初生叶尖端的较成熟细胞中发生。Norflurazon 对光敏色素积累的抑制作用在叶尖处比在茎基部处更为明显。在 48 小时黑暗期结束时，对燕麦幼苗进行 5 分钟的红光照射，导致来自绿光和 Norflurazon 漂白的燕麦茎粗提物中的光敏色素含量随后大量减少。这些观察结果排除了检测到大量无生色团的光敏色素积累的可能性，并表明 Norflurazon 在长时间黑暗期间对光敏色素积累有重大影响。
