Molecular cloning of cDNA for Avena phytochrome

Abstract

We have isolated several cDNA clones for phytochrome, a plant regulatory photoreceptor. A cDNA library was constructed by using etiolated Avena poly(A)+ RNA enriched for phytochrome mRNA by size fractionation. Replicate arrays of colonies were differentially screened with cDNA probes made from poly(A)+ RNA that had been either enriched in or depleted of phytochrome mRNA. Of the colonies hybridizing preferentially with the enriched probe, several contained plasmids that specifically selected phytochrome mRNA when assayed by hybridization-selection and translation. The largest such plasmid, pAP-2, was used to isolate clones from an Avena genomic library. One of these genomic clones was then used to screen a second cDNA library in an attempt to identify full-length phytochrome clones. The largest of the plasmids thus obtained, pAP-3, contains a 3.4-kilobasepair (kbp) insert, verified to contain phytochrome sequences by hybridization-selection and translation. Sequence analysis of pAP-2 and pAP-3 revealed that the two clones are identical in sequence through a 2.4-kbp region in which they overlap. However, the pAP-2 insert contains, in addition, 1.5 kbp of sequence of unknown origin, the apparent result of a recombination event. Blots of poly(A)+ RNA hybridized with 32P-labeled pAP-2 or pAP-3 show a single mRNA band at 4.2 kilobases. Blot analysis of RNA from dark-grown and from red-irradiated tissue demonstrates that a previously reported light-induced decrease in translatable phytochrome mRNA results from a decrease in physical abundance of this mRNA.Phytochrome is the best characterized of the regulatory photoreceptors that control plant development in response to light (1). The molecule is a chromoprotein consisting of a linear tetrapyrrole chromophore covalently linked to a polypeptide of molecular mass in the range of 120-127 kilodaltons (kDa), depending on plant species (2,3

Structure and Expression of Maize Phytochrome Family HomeologsSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY234825, AY234826, AY234827, AY234828, AY234829, AY234830.

“…The organ-and tissuespecific expression profiles of PHYB genes in tomato relative to white light in blade, sheath, and mesocotyl tissues. This light-dependent decrease in phyA tran- (Hauser et al 1997) and tobacco (Adam et al 1996) and maize (this study) contrast with the relatively uniform scripts has been observed in several grass species (Colbert et al 1983;Hershey et al 1984; Christensen and pattern of PHYB expression in Arabidopsis (Somers and Quail 1995b) and potato (Heyer and Gatz 1992) and Quail 1989;Kay et al 1989;Dehesh et al 1991) and likely reflects a common regulatory mechanism that acts suggest that there may be little conservation in the transcriptional regulation of PHYB and PHYC accumulation to maintain high levels of phyA transcript in the dark (Bruce et al 1989). Furthermore, several elements have between monocots and dicots.…”

Oat phytochrome A mRNA degradation appears to occur via two distinct pathways.

“…Interestingly, the minimum PHYA tail length in poly(A)+ RNA (-24 nucleotides) was similar to the minimum size to which the poly(A) binding protein (PABP) will associate (Baker, 1993). Previously, the oat fulllength PHYA mRNA was estimated to be 4.2 kb in size (Hershey et al, 1984). However, from the RNase H poly(A) tail data and the known length of PHYA cDNA clones (Hershey et al, 1985), the size of the polyadenylated full-length PHYA mRNA is calculated to be 4030 nucleotides.…”

Molecular cloning and sequence of cDNA encoding the plasma membrane proton pump (H+-ATPase) of Arabidopsis thaliana.

“…An Avena sativa cDNA library with G+C-tailed inserts harbored in pBR322 was generously provided by J. Lissemore and P. Quail (13 Approximately 100,000 plaques of an A. thaliana cDNA phage library were screened with a 0.6-kb fragment that was gel-purified from the oat clone pTS.6. Two independent clones, pAHA1-11 and pAHA2, were obtained.…”

“…The plasma membrane proton pump has only been observed in plants and fungi and was characterized by biochemical and electrophysiological techniques (1,8 An Avena sativa cDNA library with G+C-tailed inserts harbored in pBR322 was generously provided by J. Lissemore and P. Quail (13). Colony hybridizations were performed at 30°C in 6x SSC (lx = 0.15 M NaCl/0.015 M sodium citrate, pH 7.0)/i x Denhardt's solution (lx = 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine serum albumin)/0.05% (wt/vol) sodium pyrophosphate/yeast tRNA at 100 ,ug/ml/20% (vol/vol) formamide containing 1.1-2.2 x 107 dpm of 32P-labeled GDGV26I per ml.…”