Regulation of phytochrome mRNA abundance in green oat leaves

Abstract. Avena sativa L. (oat) seedings were grown 4 d in continuous white light followed by 3 d in darkness. Probes derived from an oat phytochrome cDNA clone (pAP 3.2) were used in slot blot analyses to measure the abundance of phytochrome mRNA in the distinct etiolated and green portions of the leaves produced by these seedlings. Both the green and etiolated portions accumulated phytochrome mRNA to a level of about 85% of the etiolated seedling level. Subsequent experiments with similar seedlings showed that both the green and etiolated portions were capable of inducing a dramatic decline in phytochrome mRNA abundance in response to a saturating red light pulse. Despite the ability of green portions of oat leaves to accumulate phytochrome mRNA and to down-regulate phytochrome mRNA abundance in response to light, no substantial variation in phytochrome mRNA abundance was observed in green oat seedlings maintained on a 12-h day/12-h night cycle. In the same oat seedlings, the abundance of chlorophyll a/b binding protein mRNA fluctuated dramatically during the day/night cycle.     

Down-regulation of phytochrome mRNA abundance by red light and benzyladenine in etiolated cucumber cotyledons

Northern blot analysis revealed that a single 4.2 kb phytochrome mRNA species was detectable in cotyledons excised from five-day-old etiolated cucumber seedlings. Intact etiolated five-day-old cucumber seedlings were given a red light or benzyladenine treatment, and cotyledons were harvested at various times following treatment. The abundance of phytochrome mRNA in the cotyledons was quantitated using ³²P-labeled RNA probes and slot blot analysis. By 2 h after irradiation the phytochrome mRNA level was reduced to 40% of the initial abundance and reaccumulation began by 3 h after irradiation. Reaccumulation of phytochrome mRNA to the time-zero dark control level was achieved by 10 h after treatment. A decrease in phytochrome mRNA abundance was evident by 2 h after benzyladenine treatment, and a maximal reduction to 45% of the time-zero dark control was attained by 4 h after treatment. No recovery of the phytochrome mRNA level was evident by 8 h after benzyladenine treatment. The abundance of actin mRNA was unaffected by benzyladenine treatment.     

CG-1, a parsley light-induced DNA-binding protein

A partial cDNA clone (CG-1) encoding a sequence-specific DNA-binding protein (CG-1) was isolated from a parsley (Petroselinum crispum L.) cDNA expression library by a DNA-ligand screening. The nucleotide sequence CGCG is a required motif in this protein's binding site. Interestingly, the mRNA coding for CG-1 accumulated rapidly and transiently in parsley cultured cells after treatment with UV-containing white light. Although the target gene(s) for CG-1 has not been identified, its sequence-specific DNA binding and expression pattern, make CG-1 a possible member of a light signal transduction chain in parsley.     

Both phytochrome A and phytochrome B are required for the normal expression of phototropism in Arabidopsis thaliana seedlings

The role of phytochrome A (phyA) and phytochrome B (phyB) in phototropism was investigated by using the phytochrome-deficient mutants phyA-101 , phyB-1 and a phyA/phyB double mutant. The red-light-induced enhancement of phototropism, which is normally observed in wild-type seedlings, could not be detected in the phyA/phyB mutant at fluences of red light between 0.1 and 19 000 μmol m⁻². The loss of phyB has been shown to have no apparent effect on enhancement, while the loss of phyA resulted in a loss of enhancement only in the low fluence range (Janoudi et al. 1997). The conclusions of the aforementioned study can now be modified based on the current results which indicate that phototropic enhancement in the high fluence range is mediated by either phyA or phyB, and that other phytochromes have no role in enhancement. First positive phototropism was unaffected in phyA-101 and phyB-1 However, the magnitude of first positive phototropism in the phyA/phyB mutant was significantly lower than that of the wild-type Landsberg parent. Thus, the presence of either phyA or phyB is required for normal expression of first positive phototropism. The time threshold for second positive phototropism is unaltered in the phyA-101 and phyB mutants. However, the time threshold in the phyA/phyB mutant is about 2 h, approximately six times that of the wild type. Finally, the magnitude of second positive phototropism in both phyA-101 and phyB-1 is diminished in comparison with the wild-type response. Thus, phyA and phyB, acting independently or in combination, regulate the magnitude of phototropic curvature and the time threshold for second positive phototropism. We conclude that the presence of phyA and phyB is required, but not sufficient, for the expression of normal phototropism.     

Opposing roles of phytochrome A and phytochrome B in early cryptochrome-mediated growth inhibition

The cryptochrome 1 (cry1) photoreceptor is responsible for the majority of the inhibitory effect of blue light on hypocotyl elongation, but phytochrome photoreceptors also contribute to the response through a phenomenon known as coaction. In Arabidopsis thaliana the participation of phytochromes A and B (phyA and phyB) in the early phase of cry1 action was investigated by determining the effects of phyA, phyB and hy1 mutations on a cry1-dependent membrane depolarization, which is caused by the activation of plasma-membrane anion channels within seconds of blue light treatment. High-resolution growth measurements were also performed to determine the timing of the requirement for phytochrome in cry1-mediated growth inhibition, which is causally linked to the preceding anion-channel activation. A null mutation in PHYA impaired the membrane depolarization and prevented the early cry1-dependent phase of growth inhibition as effectively and with the same time course as mutations in CRY1. Thus, phyA is necessary for cry1/cry2 to activate anion channels within the first few seconds of blue light and to suppress hypocotyl elongation for at least 120 min. This finding furthers the notion of an intimate mechanistic association between the cry and phy receptors in mediating light responses. The absence of phyB did not affect the depolarization or growth inhibition during this time frame. Instead, double mutant analyses showed that the phyB mutation suppressed the early growth phenotypes of both phyA and cry1 seedlings. This result is consistent with the emerging view that the prevailing growth rate of a stem is a compromise between light-dependent inhibitory and promotive influences. It appears that phyB opposes the cry1/phyA-mediated inhibition by promoting growth during at least the first 120 min of blue light treatment.     

Impaired stem-growth responses to blue-light irradiance in light-grown transgenic tobacco seedlings overexpressing Avena phytochrome A

The effects of blue light (B) on stem extension-growth were compared in light-grown seedlings, of tobacco overexpressing Avena phytochrome A and its isogenic wild type (WT). Under natural radiation, lowering the levels of B reaching the whole shoot promoted stem extension growth in WT but not in transgenic seedlings. Under controlled conditions, the seedlings were exposed to white light (WL) or WL minus B, each one provided at two different irradiances. In WT seedlings stem extension growth was promoted by lowering B at both irradiance levels. In transgenic seedlings a reduction of B was promotive only at low irradiance levels. The seedlings were also grown under WL, WL minus B, WL minus red light (R) and far-red light (FR) or WL minus R, FR and B. In the WT, lowering B promoted stem extension growth irrespective of R+FR levels. In the transgenics, B was effective only at very low levels of R+FR (i.e. at low phytochrome cycling rates). Lowering the Pfr levels at the end of the day promoted extension growth in wild type and transgenic seedlings. Responses to B were not observed in transgenic seedlings having low Pfr levels at the end of the day. The results suggest that the overexpressed phytochrome A acts mainly via irradiance-dependent reactions. When these reactions are highly expressed, B responses are not observed.     

Tissue‐specific and light‐dependent regulation of phytochrome gene expression in rice

Phytochromes are red‐ and far red light photoreceptors in higher plants. Rice (Oryza sativa L.) has three phytochromes (phyA, phyB and phyC), which play distinct as well as cooperative roles in light perception. To gain a better understanding of individual phytochrome functions in rice, expression patterns of three phytochrome genes were characterized using promoter‐GUS fusion constructs. The phytochrome genes PHYA and PHYB showed distinct patterns of tissue‐ and developmental stage‐specific expression in rice. The PHYA promoter‐GUS was expressed in all leaf tissues in etiolated seedlings, while its expression was restricted to vascular bundles in expanded leaves of light‐grown seedlings. These observations suggest that light represses the expression of the PHYA gene in all cells except vascular bundle cells in rice seedlings. Red light was effective, but far red light was ineffective in gene repression, and red light‐induced repression was not observed in phyB mutants. These results indicate that phyB is involved in light‐dependent and tissue‐specific repression of the PHYA gene in rice.     

Nitrate reductase in cotyledons of cucumber seedlings as affected by nitrate, phytochrome and calcium

Regulation by the active form of phytochrome (P_FR) and the effect of Ca²⁺ was examined with nitrate reductase (NR) in etiolated cucumber ( Cucumis sativus cv. Beilpuig). Nitrate reductase activity (NRA) was studied in excised cotyledons of cucumber seedlings grown in distilled water and in darkness for seven days at 24 ± 0.5°C. All experiments were performed in the dark and a dim green safelight was used during analyses. In etiolated cucumber cotyledons NRA was induced by nitrate and a brief irradiation (15 min) with red light (R) resulted in 62% increase in NRA. This effect was nullified when R was followed immediately by a brief (5 min) far-red light (FR). NRA also showed a semidian (12 h) rhythmicity. Both P_FR, and nitrate effects were age dependent. Calcium seemed to be involved since the phytochrome effect was only observed when calcium was supplied in the external solution. The effect of R on NRA depended on the period of calcium nitrate incubation. An external supply of calcium ionophore mimicked the effect of R and, if supplied to R-irradiated cotyledons, produced a higher NR level than that caused by R alone. This suggested that intracellular free calcium was involved.     

Distribution of phytochrome and chlorophyll-a/b-binding-protein mRNAs in etiolated Avena seedlings

In 4-d-old dark-grown oat (Avena sativa L.) seedlings, the majority of the type-I-phytochrome (phyA) mRNA was found within 10 mm of the tip of the coleoptile sheath and in the mesocotyl node; almost none was detected in the enclosed primary leaf. In contrast, chlorophyll-a/b-binding-protein (cab) mRNAs were found almost exclusively in the enclosed primary leaf and were barely detectable in total-RNA samples from the coleoptile sheath or mesocotyl node of red-light-treated etiolated seedlings. Separated, dark-grown primary leaves responded to a red-light treatment by increasing cab-mRNA abundance in the absence of the coleoptile sheath or mesocotyl node tissues.Abbreviations cab gene for chlorophyll-a/b-binding protein - kb kilobase - phyA gene for type-I-phytochrome protein We are grateful to the members of the laboratory Dave Higgs, Theresa Tirimanne, Dr. Dennis Byrne, Bruce Held, Linda Barnes, Dr. Isaac John, and Iffat Rahim, for their helpful discussions and critical review. This work was supported by USDA grant No. 88-37261-4196 and No. 91-37304-6397, the Iowa State University Biotechnology Program, and the Molecular, Cellular, and Developmental Biology Program.     

Phytochrome A, phytochrome B and other phytochrome(s) regulate ATHB-2 gene expression in etiolated and green Arabidopsis plants

The expression of the Arabidopsis ATHB-2 gene is light-regulated both in seedlings and in adult plants. The gene is expressed at high levels in rapidly elongating etiolated seedlings and is down-regulated by a pulse of red light (R) through the action of a phytochrome other than phytochrome A or B, or by a pulse of far-red light (FR) through the action of phytochrome A. In green plants, the expression of the ATHB-2 gene is rapidly and strongly enhanced by lowering the R:FR ratio perceived by a phytochrome other than A or B. Returning the plant to a high R:FR ratio results in an equally rapid decrease of the ATHB-2 mRNA. Consistently, plants overproducing ATHB-2 show developmental phenotypes characteristic of plants grown in low R:FR: elongated petioles, reduced leaf area, early flowering, and reduced number of rosette leaves. Taken together, the data strongly suggest a direct involvement of ATHB-2 in light-regulated growth phenomena throughout Arabidopsis development.     

Intracellular redistribution of phytochrome in etiolated soybean (Glycine max L.) seedlings

The intracellular distribution of phytochrome in hypocotyl hooks of etiolated soybean (Glycine max L.) has been examined by immunofluorescence using a newly produced monoclonal antibody (Soy-1) directed to phytochrome purified from etiolated soybean shoots. Cortical cells in the hook region exhibit the strongest phytochrome-associated fluorescence, which is diffusely distributed throughout the cytosol in unirradiated, etiolated seedlings. A redistribution of immunocytochemically detectable hytochrome to discrete areas (sequestering) following irradiation with red light requires a few minutes at room temperature in soybean, whereas this redistribution is reversed rapidly following irradiation with far-red light. In contrast, sequestering in oat (Avena sativa L.) occurs within a few seconds (D. McCurdy and L. Pratt, 1986, Planta 167, 330–336) while its reversal by far-red light requires hours (J. M. Mackenzie Jr. et al., 1975, Proc. Natl. Acad. Sci. USA 72, 799–803). The time courses, however, of red-light-enhanced phytochrome pelletability and sequestering are similar for soybean as they are for oat. Thus, while these observations made with a dicotyledon are consistent with the previous conclusion derived from work with oat, namely that sequestering and enhanced pelletability are different manifestations of the same intracellular event, they are inconsistent with the hypothesis that either is a primary step in the mode of action of phytochrome.Abbreviations DIC differential interference contrast - FR far-red light - Ig immunoglobulin - Pfr, P far-red- and red-absorbing form of phytochrome, respectively - R red light This work was supported by National Science Foundation grant No. DCB-8703057.