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A light‐ and temperature‐entrained circadian clock controls expression of transcripts encoding nuclear proteins with homology to RNA‐binding proteins in meristematic tissue
Author(s) -
Heintzen Christian,
Melzer Slegbert,
Fischer Ruth,
Kappeler Stefan,
Apel Klaus,
Staiger Dorothee
Publication year - 1994
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.1994.5060799.x
Subject(s) - biology , zeitgeber , circadian clock , circadian rhythm , complementary dna , sinapis , meristem , northern blot , messenger rna , microbiology and biotechnology , rna , gene expression , ultradian rhythm , botany , gene , genetics , endocrinology , shoot , brassica
Summary 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 aiba 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, Sa grp1 and Sa grp2, 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 ‘Zeitgober’ 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 Sa grp 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 A1 of the heterogeneous nuclear ribonucleoprotein complex involved in pre‐mRNA splicing. The Sa GRP protein fluctuates with a very low amplitude over light/dark cycles. Immunogold labeling demonstrates the presence of the Sa GRP protein within the nucleus of the investigated meristematic cells of young leaves.