A LIGHT-ENTRAINED AND TEMPERATURE-ENTRAINED CIRCADIAN CLOCK CONTROLS EXPRESSION OF TRANSCRIPTS ENCODING NUCLEAR PROTEINS WITH HOMOLOGY TO RNA-BINDING PROTEINS IN MERISTEMATIC TISSUE
A LIGHT-ENTRAINED AND TEMPERATURE-ENTRAINED CIRCADIAN CLOCK CONTROLS EXPRESSION OF TRANSCRIPTS ENCODING NUCLEAR PROTEINS WITH HOMOLOGY TO RNA-BINDING PROTEINS IN MERISTEMATIC TISSUE
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DOI：
10.1046/j.1365-313x.1994.5060799.x
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发表时间：
1994-06-01
期刊：
影响因子：
7.2
通讯作者：
STAIGER, D
中科院分区：
文献类型：
--
作者：
HEINTZEN, C;MELZER, S;STAIGER, D
为了在分子水平上研究内源性昼夜节律产生的生理过程，我们在长日照植物白芥中鉴定了时钟控制基因。使用代表在光周期中间或暗周期中间表达的转录物的cDNA探针差异筛选cDNA文库。两组密切相关的成绩单，Sagrp 1和Sagrp 2，控制的昼夜节律已被隔离。RNA印迹分析证实，在光/暗循环中生长的植物中，转录水平振荡，最大值在“Zeitgeber”时间（zt）8和zt 12（光照开始后8-12小时）之间，最小值在zt 20左右。稳态mRNA水平在从光/暗周期转移到恒定光照的植物中继续振荡。没有同步的mRNA振荡被发现在植物生长的种子在恒定的光照和恒定的温度，这表明时钟必须被夹带最初。与此相反，当植物生长在恒定的光暴露于有节奏的温度变化振荡的稳态Sagrp mRNA水平的诱导，表明温度作为一种替代的外部刺激（zeitgeber）以外的光夹带的振荡器。原位杂交表明，这两个转录组主要在分生组织和生长组织中表达。在茎尖叶原基、原形成层束、形成层和zt 12左右幼叶的所有细胞层中观察到强表达。相反，在zt 20分离的组织切片上发现很少或没有信号。这表明调节Sagrp转录物波动的振荡器在不同器官中同步操作。对于两个转录物组，分离对应于未剪接的前mRNA或对应于通过使用第二个5 ′剪接位点产生的转录物的cDNA。对应于完全剪接的转录物的cDNA含有16 kDa多肽的开放阅读框，每个多肽含有推定的N-末端RNA识别基序和富含甘氨酸的C-末端区域。预测的蛋白质显示出很强的同源性ABA诱导的富含甘氨酸的蛋白质从玉米胚和哺乳动物RNA结合蛋白Al的异质性核核糖核蛋白复合物参与前mRNA剪接。SaGRP蛋白在光/暗循环中以非常低的幅度波动。免疫金标记证明了在所研究的幼叶分生组织细胞的细胞核内存在SaGRP蛋白。
To investigate physiological processes generated by endogenous circadian rhythms on the molecular level, we have identified clock-controlled genes in the long-day plant Sinapis alba L. A cDNA library was differentially screened using cDNA probes representing transcripts expressed at either the middle of the light period or the middle of the dark period. Two closely related groups of transcripts, Sagrp1 and Sagrp2, controlled by a circadian rhythm have been isolated. RNA blot analysis verified that transcript levels oscillate in plants grown in light/dark cycles with maxima between 'Zeitgeber' time (zt)8 and zt12 (8-12 h after onset of illumination) and minima around zt20. Steady-state mRNA levels continue to oscillate in plants shifted from light/dark cycles to constant light. No synchronous mRNA oscillations are found in plants grown from seed in constant light at constant temperature, suggesting that the clock has to be entrained initially. In contrast, when plants grown in constant light are exposed to rhythmic temperature shifts oscillations of steady-state Sagrp mRNA levels are induced, indicating that temperature acts as an alternative external stimulus (zeitgeber) other than light to entrain the oscillator. In situ hybridization reveals that both transcript groups are expressed predominantly in meristematic and growing tissue. Strong expression is observed in the leaf primordia of the shoot apex, the procambial strands, cambium and in all cell layers of young leaves around zt12. In contrast, little or no signal is found on tissue sections isolated at zt20. This indicates that the oscillator(s) regulating Sagrp transcript fluctuations operate(s) synchronously in different organs. for both transcript groups cDNAs were isolated corresponding to unspliced pre-mRNAs or to transcripts generated by the use of a second 5' splice site. The cDNAs corresponding to the fully spliced transcripts contain open reading frames for polypeptides of 16 kDa, each containing a putative N-terminal RNA recognition motif and a C-terminal region rich in glycine. The predicted proteins show strong homology to an ABA-inducible glycine-rich protein from maize embryos and to the mammalian RNA-binding protein Al of the heterogeneous nuclear ribonucleoprotein complex involved in pre-mRNA splicing. The SaGRP protein fluctuates with a very low amplitude over light/dark cycles. Immunogold labeling demonstrates the presence of the SaGRP protein within the nucleus of the investigated meristematic cells of young leaves.