Using four o'clock as a model system to study petal senescence.

摘要

As a preliminary step to develop a transformation protocol for four o'clock (Mirabilis jalapa L.), the chosen model species for studying petal senescence, a regeneration protocol was developed. The cotyledons and their subtending petioles were isolated from mature seeds and cultured in the dark for one week on Murashige and Skoog (MS) medium containing 1mg·L -1 IAA and 1mg·L-1 thiadiazuron (TDZ). The cotyledons were then transferred to shoot induction media (MS salts and vitamins, 2 mg·L-1 TDZ) and cultured in the light. After four weeks, shoot meristems appeared on more than 80% of the petioles. Most of the meristems could develop to mature shoots, and then to intact plants. The time of synthesizing transcripts regulating four o'clock flower senescence was determined by alpha-amanitin treatment. Genes differentially expressed before and after this time were isolated using PCR-assisted subtractive hybridization. Transcripts of 82 up-regulated sequences and 65 down-regulated sequences were identified. They included several transcription factors, including a bZIP transcription factor and a homeodomain-leucine zipper (HD-Zip) transcription factor in the up-regulated library and two Myb transcription factors in the down-regulated library. Expression patterns of these and other selected genes during four o'clock flower opening and senescence were examined by real time PCR. The possible roles in flower senescence of these transcription factors and other intriguing sequences are discussed. The promoter of MjXB3 , a gene encoding a RING zinc finger ankyrin repeat protein whose expression increased 40,000 fold in the initiation of four o'clock flower senescence, was isolated. Database search revealed that the promoter contains binding sites for several kinds of transcription factors, including bZIP, HD-Zip, Myb, MADS-box and WRKY transcription factors. A lkb fragment immediate upstream the MjXB3 gene was sufficient to drive GUS transient expression specifically in senescing petunia flowers. It also functioned specifically in senescing carnation flowers. However, this promoter could not drive GUS expression in senesced flowers of daffodil, daylily or orchid, which are monocotyledons. The possible role of the RING zinc finger gene in flower senescence and the possible application of its promoter in biotechnological modification of flower longevity were discussed.