Cloning of cDNA for phytochrome from etiolated Cucurbita and coordinate photoregulation of the abundance of two distinct phytochrome transcripts

Abstract

We have isolated several cDNA clones for phytochrome from a dicot, Cucurbita pepo L. cv. Black Beauty (zucchini), and have used them to study the regulation of Cucurbita phytochrome mRNA levels. A cDNA library was constructed from poly(A)(+) RNA isolated from etiolated Cucurbita hypocotyl hooks and enriched for phytochrome mRNA by size fractionation. This library was screened with a (32)P-labeled fragment isolated from an Avena phytochrome cDNA clone. Several putative phytochrome clones were isolated and mapped by restriction endonuclease analysis. On the basis of this analysis there is no evidence for the expression of multiple phytochrome genes in Cucurbita. Recent sequence analysis has confirmed that the largest of these clones, pFMD1 (∼3.6 kb), does indeed encode phytochrome and that it contains the entire amino acid coding sequence for Cucurbita phytochrome (33). RNA blot analysis has revealed that two polyadenylated phytochrome transcripts (∼5.6 kb and ∼4.2 kb) are present in both cotyledons and hypocotyl hooks of Cucurbita. In etiolated Cucurbita seedlings given a saturating pulse of red light, the abundance of both transcripts coordinately declines to 50-60% of the dark levels within 3 h and reaccumulates to dark levels within 24 h. Reversal of induction of this response by a far-red light pulse immediately following red light treatment is not observed, which is in contrast to the far-red reversibility of the red light promoted decrease in phytochrome mRNA abundance observed in Avena (6). Etiolated seedlings transferred to continuous white light also show a coordinate decrease in the levels of the two RNAs to ∼40% of the dark levels within 3 h. The magnitude of the light-induced decline in phytochrome mRNA abundance in Cucurbita is substantially less than the decrease previously reported for Avena (6).

Regulation of Phytochrome Message Abundance in Root Caps of Maize

“…The magnitude of the previously observed decreases in message levels ranged from 10-to 20-fold reductions in oats (3) and pea (19) to approximately 5-fold in rice (10) and only a 2-fold decrease in zucchini ( 12). In each of these cases, the samples examined included etiolated shoot tissue.…”

“…Furthermore, this regulation results in an observed decrease in the abundance of phytochrome message due, at least in part, to a reduction in transcriptional activity (13). The response has been observed in maize seedlings (2), although to a lesser extent than seen in other species, such as oat (3), rice (10), and pea (26), and to a greater extent than in zucchini (12). In these studies organs such as etiolated coleoptiles or cotyledons that accumulate relatively high levels of phytochrome message in the dark have been examined; there is little information (10) on the abundance of phytochrome message in root tissues.…”

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

“…Total RNA was isolated with a small-scale RNA isolation procedure (Seeley et al, 1992). Poly(A)+ and poly(A)-RNA samples were selected with a poly(U) Sephadex column (Bio-Rad) (Murray et al, 1981;Lissemore et al, 1987) from total RNA and prepared by a large-scale SDS-phenol procedure (Dean et al, 1985). Polysomal RNA was isolated as described by Byrne et al (1993).…”

“…This labeled, GUS mRNA was added to 5 mg of SDS-phenol-extracted oat total RNA prior to poly(A) selection (Lissemore et al, 1987).…”

Characterization of Tobacco Expressing Functional Oat Phytochrome

“…This is accomplished through mechanisms that control: (a) the transcription of phytochrome genes, (b) phytochrome mRNA turnover, and (c) selective degradation of Pfr (4,33). In the monocots and to a much lesser extent in the dicots examined, abundance of phytochrome mRNA decreases upon photoconverison of Pr to Pfr (4,21). This decline is rapid, occurring within minutes after Pfr formation, and has been shown to involve transcriptional attenuation.…”