A LIGHT-ENTRAINED AND TEMPERATURE-ENTRAINED CIRCADIAN CLOCK CONTROLS EXPRESSION OF TRANSCRIPTS ENCODING NUCLEAR PROTEINS WITH HOMOLOGY TO RNA-BINDING PROTEINS IN MERISTEMATIC TISSUE

To investigate physiological processes generated by endogenous circadi an rhythms on the molecular level, we have identified clock-controlled genes in the long-day plant Sinapis alba L. A cDNA library was differ entially screened using cDNA probes representing transcripts expressed at either the middle of the light period or the middle of the dark pe riod. Two closely related groups of transcripts, Sagrp1 and Sagrp2, co ntrolled by a circadian rhythm have been isolated. RNA blot analysis v erified that transcript levels oscillate in plants grown in light/dark cycles with maxima between 'Zeitgeber' time (zt)8 and zt12 (8-12 h af ter onset of illumination) and minima around zt20. Steady-state mRNA l evels continue to oscillate in plants shifted from light/dark cycles t o 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 plant s grown in constant light are exposed to rhythmic temperature shifts o scillations 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 reve als that both transcript groups are expressed predominantly in meriste matic and growing tissue. Strong expression is observed in the leaf pr imordia of the shoot apex, the procambial strands, cambium and in all cell layers of young leaves around zt12. In contrast, little or no sig nal 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 we re isolated corresponding to unspliced pre-mRNAs or to transcripts gen erated by the use of a second 5' splice site. The cDNAs corresponding to the fully spliced transcripts contain open reading frames for polyp eptides of 16 kDa, each containing a putative N-terminal RNA recogniti on motif and a C-terminal region rich in glycine. The predicted protei ns show strong homology to an ABA-inducible glycine-rich protein from maize embryos and to the mammalian RNA-binding protein Al of the heter ogeneous nuclear ribonucleoprotein complex involved in pre-mRNA splici ng. The SaGRP protein fluctuates with a very low amplitude over light/ dark cycles. Immunogold labeling demonstrates the presence of the SaGR P protein within the nucleus of the investigated meristematic cells of young leaves.