The interaction between cold and light controls the expression of the cold-regulated barley gene cor14b and the accumulation of the corresponding protein

Abstract

We report the expression of the barley (Hordeum vulgare L.) COR (cold-regulated) gene cor14b (formerly pt59) and the accumulation of its chloroplast-localized protein product. A polyclonal antibody raised against the cor14b-encoded protein detected two chloroplast COR proteins: COR14a and COR14b. N-terminal sequencing of COR14a and expression of cor14b in Arabidopsis plants showed that COR14a is not encoded by the cor14b sequence, but it shared homology with the wheat (Triticum aestivum L.) WCS19 COR protein. The expression of cor14b was strongly impaired in the barley albino mutant an, suggesting the involvement of a plastidial factor in the control of gene expression. Low-level accumulation of COR14b was induced by cold treatment in etiolated plants, although cor14b expression and protein accumulation were enhanced after a short light pulse. Light quality was a determining factor in regulating gene expression: red or blue but not far-red or green light pulses were able to promote COR14b accumulation in etiolated plants, suggesting that phytochrome and blue light photoreceptors may be involved in the control of cor14b gene expression. Maximum accumulation of COR14b was reached only when plants were grown and/or hardened under the standard photoperiod. The effect of light on the COR14b stability was demonstrated by using transgenic Arabidopsis. These plants constitutively expressed cor14b mRNAs regardless of temperature and light conditions; nevertheless, green plants accumulated about twice as much COR14b protein as etiolated plants.

Figure 3

Light-dependent accumulation of COR14. a, Barley plants (cv Onice) were grown and hardened for 7 d under different light conditions. Total protein extracts were separated by Tricine-SDS-PAGE and hybridized with COR14 antibody; the 29-kD anonymous protein band used for western blot normalization is also indicated. Lane 1, Green plants grown at 20°C; lane 2, green plants grown and hardened under the standard photoperiod; lane 3, etiolated plants hardened under the standard photoperiod; lane 4, green plants hardened in the dark; lane 5, etiolated plants hardened in the dark; and lane 6, etiolated plants exposed for 5 min to light (200 μmol m⁻² s⁻¹) and then hardened in the dark. b, Etiolated barley plants (cv Onice) were hardened in the dark for 13 d and compared with plants grown and hardened in the same conditions, except for a pulse of white light (5 min at 200 μmol m⁻² s⁻¹) after 7 d of hardening or before hardening. Total protein extracts were separated by Tricine-SDS-PAGE and hybridized with COR14 antibody. Lane 1, Green plants hardened under the standard photoperiod; lane 2, etiolated plants hardened in the dark for 8 d; lane 3, etiolated plants hardened in the dark for 10 d; lane 4, etiolated plants hardened in the dark for 13 d; lane 5, etiolated plants hardened in the dark for 7 d, exposed to light, and further hardened in the dark for 1 d; lane 6, etiolated plants hardened in the dark for 7 d, exposed to light, and further hardened in the dark for 3 d; lane 7, etiolated plants hardened in the dark for 7 d, exposed to light, and further hardened in the dark for 6 d; lane 8, etiolated plants exposed to light before 1 d of hardening in the dark; lane 9, etiolated plants exposed to light before 3 d of hardening in the dark; and lane 10, etiolated plants exposed to light before 6 d of hardening in the dark.

Figure 6

Red (660 nm) and blue (400 nm) light were the most effective in stimulating COR14b accumulation. Etiolated barley plants (cv Onice) were exposed to 5 min of different light spectra (about 10 μmol m⁻² s⁻¹) and then hardened for 7 d in the dark. a, Result obtained after about 10 s of autoradiography exposure so that only the strongest signals were recorded. b, A 30-s exposure of the same western blot is presented to allow the detection of a basal level of COR14b induced by far-red, green, and 450 nm of light, whereas the effect of the cold signal alone was even lower (see also c, no light sample). Total protein extracts were separated by Tricine-SDS-PAGE and hybridized with COR14 antibody. Lane 1, Green plants hardened under the standard photoperiod; lane 2, etiolated plants hardened in the dark after 5 min of white light; lane 3, etiolated plants hardened in the dark after 5 min of red (660 nm) light; lane 4, etiolated plants hardened in the dark after 5 min of far-red (730 nm) light; lane 5, etiolated plants hardened in the dark after 5 min of red (660 nm) plus 5 min of far red (730 nm) light; lane 6, etiolated plants hardened in the dark after 5 min of green (500 nm) light; lane 7, etiolated plants hardened in the dark after 5 min of blue (450 nm) light; lane 8, etiolated plants hardened in the dark after 5 min of blue (400 nm) light; and lane 9, etiolated plants hardened in the dark. c, Etiolated barley plants (cv Onice) were exposed to a short period of red light (200 μmol m⁻² s⁻¹) and then hardened for 7 d in the dark. Total protein extracts were separated by Tricine-SDS-PAGE and hybridized with COR14 antibody. Lane 1, Green plants grown at 20°C; lane 2, green plants hardened under the standard photoperiod; lane 3, etiolated plants hardened in the dark after 5 min of light; lane 4, etiolated plants hardened in the dark after 3 min of light; lane 5, etiolated plants hardened in the dark after 2 min of light; lane 6, etiolated plants hardened in the dark after 1 min of light; lane 7, etiolated plants hardened in the dark after 30 s of light; lane 8, etiolated plants hardened in the dark after 15 s of light; lane 9, etiolated plants hardened in the dark after 5 s of light; and lane 10, etiolated plants hardened in the dark.

Figure 4

Albino plants show a minimal cor14b expression. Barley plants carrying the homozygote albino mutation a_n, the corresponding heterozygotes, and plants of the barley cv Onice were hardened for 7 d under different light conditions. A Northern blot made with poly(A⁺) RNAs was probed with cor14b (a) and normalized with [³²P]dATP-labeled oligo(dT) (b). Total protein extracts were separated by Tricine-SDS-PAGE and hybridized with COR14 antibody (c). Lane 1, Green plants grown at 20°C; lane 2, albino mutant a_n grown at 20°C; lane 3, green plants (cv Onice) hardened under the standard photoperiod; lane 4, albino mutant a_n hardened under the standard photoperiod; lane 5, etiolated plants hardened in the dark; and lane 6, green heterozygote a_n plants hardened under the standard photoperiod.

Figure 7

Etiolated plants were exposed to white light for 5 min and kept in the dark at 20°C for a few days before 7 d of hardening in the dark. Total protein extracts were separated by Tricine-SDS-PAGE and hybridized with COR14 antibody (a). A northern blot made with poly(A⁺) RNAs was probed with the COR genes cor14b (b) and paf93 (c). Lane 1, Green plants hardened under the standard photoperiod; lane 2, 7-d-old etiolated plants exposed to light before hardening in the dark; lane 3, 7-d-old etiolated plants exposed to light, kept in the dark for 1 d, and hardened in the dark; lane 4, 7-d-old etiolated plants exposed to light, kept in the dark for 2 d, and hardened in the dark; lane 5, 7-d-old etiolated plants exposed to light, kept in the dark for 3 d, and hardened in the dark; lane 6, 7-d-old etiolated plants exposed to light, kept in the dark for 5 d, and hardened in the dark; lane 7, same as lane 5 without light treatment; lane 8, same as lane 6 without light treatment; lane 9, 10-d-old etiolated plants exposed to light and then hardened in the dark; lane 10, 12-d-old etiolated plants exposed to light and then hardened in the dark; lane 11, same as lane 8 plus 7 d of hardening under the standard photoperiod; lane 12, same as lane 8 plus 4 d at 20°C and 7 d of hardening under the standard photoperiod.

Figure 1

cor14b encodes for COR14b. a, Total protein extracts from wild-type Arabidopsis (wt), transgenic Arabidopsis containing the chimeric construct 35SCaMV-cor14b-NOS, and cold-hardened (7 d at 3°C) barley (cv Onice) were separated by Tricine-SDS-PAGE and hybridized with COR14 antibody. b, Protein extracts isolated from different compartments of barley (cv Onice) and of the transgenic Arabidopsis line C3–15 were separated by Tricine-SDS-PAGE and hybridized with COR14 antibody. Lane 1, Total protein extract from barley grown at 20°C; lane 2, total protein extract from barley hardened 7 d at 3°C; lane 3, thylakoid protein extract from barley hardened 7 d at 3°C; lane 4, stroma protein extract from barley hardened 7 d at 3°C; lane 5, total protein extract from wild-type Arabidopsis; lane 6, thylakoid protein extract from transgenic Arabidopsis; and lane 7, stroma protein extract from transgenic Arabidopsis.

Figure 2

Amino acid sequence alignment among WCS19 from wheat, COR14b, as deduced from the cDNA clones, and the N-terminal sequence of COR14a. “I” and “x” indicate a perfect match and homologous substitution, respectively, between WCS19 and COR14b. “#” and “*” indicate a perfect match and homologous substitution, respectively, between WCS19 and COR14a.

Figure 5

Accumulation of COR14b in transgenic Arabidopsis plants. Green and etiolated Arabidopsis plants transformed with the chimeric construct 35SCaMV-cor14b were analyzed for the amount of cor14b mRNA (a) and of the corresponding COR14b protein (b). A northern blot made with total RNAs was stained with ethidium bromide (c) and probed with the cDNA clone cor14b; total protein extracts were separated by Tricine-SDS-PAGE and hybridized with COR14 antibody. Lane 1, Green transgenic plants hardened under the standard photoperiod; lane 2, etiolated transgenic plants hardened in the dark; lane 3, green transgenic plants grown at 22°C; lane 4, etiolated transgenic plants grown at 22°C; and lane 5, wild-type plants grown at 22°C.