Phytochrome-mediated control of COP1 gene expression in rice plants

We isolated a COP1 cDNA from rice and found that it could complement the Arabidopsis cop1-4 mutant. The putative rice COP1 protein has the Ring-finger, coiled-coil. and WD-40 repeat domains, which are also conserved in pea, tomato, and mammalian COP1 proteins. The degree of overall identity between rice COP1 and Arabidopsis COP1 is 73%, and the similarity value is 83%. Expression of rice COP1 was detected in almost all plant tissues, with the level being relatively higher in calli and very low in etiolated leaves. The expression level was positively controlled by light in etiolated and green leaves. At the end of the light period, expression of the gene in green leaves could be down-regulated by far-red light. This far-red light effect could be prevented by subsequent irradiation with red light. These results indicate that phytochrome regulates rice COP1 expression.     

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.     

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.     

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.     

Differences in Photoresponse and Phytochrome Spectrophotometry Between Etiolated and De-etiolated Pea Stem Tissue

Morphologically similar pea plants having a 4-fold difference in spectrophoto-metrically detectable phytochrome can be produced by pretreatment of etiolated plants with red light (R) or with red and far-red light combined (RF). A search for response differences which could be ascribed to differences in phytochrome content has resulted only in the establishment of differences due to de-etiolation. Segments of etiolated plants differ from those of plants de-etiolated by R and RF pretreatments in 2 ways. Segments from etiolated plants appear to respond rapidly to the far-red absorbing form of phytochrome (P_FR), while segments from de-etiolated plants do not respond rapidly to P_FR. This statement is based upon 2 observations: (i) the red light induced growth inhibition in segments from etiolated plants rapidly escapes reversibility by far-red light, while with segments from R or RF pretreated plants, the red light effect is fully reversed by subsequent far-red light for up to 2 hr; and (ii) segments from etiolated plants were inhibited to a greater degree than were segments from RF pretreated plants when various photostationary state levels of P_FR were maintained for 30 or 90 min and then removed by photoconversion to P_R. The in vivo nonphotochemical transformation curves of the phytochrome of etiolated and RF pretreated plants appear to differ in 2 related respects: (i) the amount of phytochrome destroyed in de-etiolated tissue is greater than that in etiolated tissue, perhaps as a result of the fact that (ii) the rate and extent of apparent reversion of P_FR to P_R in etiolated tissue is about twice that in de-etiolated tissue.     

Photorespiration and light act in concert to regulate the expression of the nuclear gene for chloroplast glutamine synthetase.

In Pisum sativum, distinct chloroplast and cytosolic forms of glutamine synthetase (GS) are encoded by homologous nuclear genes that are differentially expressed in vivo (Tingey, S. V., Tsai, F.-Y., Edwards, J. W., Walker, E. L., and Coruzzi, G. M. [1988]. J. Biol. Chem. 263, 9651-9657). In leaves, light selectively affects the expression of the nuclear gene for chloroplast GS2. Differences in the maximal levels of GS2 mRNA in etiolated plants treated with red or white light indicate that only part of the white-light-induced accumulation of GS2 mRNA is due to a phytochrome-mediated response. The kinetics of GS2 mRNA accumulation in response to white-light illumination of etiolated or dark-adapted green plants indicates that GS2 mRNA accumulates more rapidly in plants containing mature, photosynthetically competent chloroplasts. Other evidence that GS2 mRNA levels are affected by the metabolic status of chloroplasts concerns the selective induction of GS2 mRNA in plants grown under conditions that result in the production of photorespiratory ammonia. These results indicate that the light-induced accumulation of GS2 mRNA in leaves results from the action of phytochrome as well as light-induced changes in chloroplast metabolism.     

Isolation and characterization of a cDNA-clone coding for potato type A phytochrome

We have isolated and sequenced a cDNA clone encoding the apoprotein of a potato phytochrome. Based on the deduced amino acid sequence, which shows 78% amino acid identity to the Arabidopsis phyA and 50% identity to the Arabidopsis phyB open reading frame, we have classified this cDNA clone as potato phyA phytochrome. The amino acid immediately preceding cysteine 323, which is the homologue of oat cystein 321, to which the chromophore has been shown to be attached, is a tyrosine residue. This contrasts with six other type A phytochrome sequences from both monocots and dicots that encode serine in this position. As already observed in three other cDNAs isolated from dicot species, the potato phyA clone encodes a short open reading frame (13 amino acids) preceding the phyA open reading frame (1123 amino acids), supporting the idea that this type of leader sequence might be involved in the regulated expression of the phytochrome apoprotein. Southern blot analysis revealed a single phyA gene as well as other related phytochrome sequences in the potato genome. phyA mRNA levels varied in different organs and were modulated by white light; in seedlings and sprouts, highest levels of mRNA were detected in the etiolated stage. Upon illumination with white light, mRNA levels decreased to the amount found in leaves of re-etiolated plants. Lowest expression was observed in leaves of plants grown in the light, in tubers irrespective of light treatment, and in roots of plants grown in the dark. In roots of plants grown in the light, elevated levels of phyA mRNA were detected. Using a monoclonal antibody generated against pea phytochrome as an immunochemical probe, the protein was only detectable in protein extracts from etiolated seedlings and sprouts.     

Light-Dependence of Phospholipase D Activity in Oat Seedlings

The effect of light on the activity of phospholipase D (PLD) in oat (Avena sativa L.) seedlings and the dependence of this enzyme activity on the regime of their illumination were studied. The PLD activity in etiolated seedlings was 1.5–2.0-fold higher than in green plants. The illumination of etiolated seedlings with white light resulted in a decrease in PLD activity to its level in the seedlings grown under light. In contrast, the transfer of green seedlings to darkness enhanced the activity of the enzyme up to its level in etiolated seedlings. The illumination of etiolated seedlings with red light inhibited the PLD as well. It was shown that this photoeffect decreased with seedling aging and correlated with a phytochrome content in plants. Far-red light reversed the effect of red light. The involvement of phytochrome in the control of the PLD activity is discussed.     

Expression of functional oat phytochrome A in transgenic rice.

To investigate the biological functions of phytochromes in monocots, we generated, by electric discharge particle bombardment, transgenic rice (Oryza sativa cv Gulfmont) that constitutively expresses the oat phytochrome A apoprotein. The introduced 124-kD polypeptide bound chromophore and assembled into a red- and far-red-light-photoreversible chromoprotein with absorbance spectra indistinguishable from those of phytochrome purified from etiolated oats. Transgenic lines expressed up to 3 and 4 times more spectrophotometrically detectable phytochrome than wild-type plants in etiolated and green seedlings, respectively. Upon photo-conversion to the far-red-absorbing form of phytochrome, oat phytochrome A was degraded in etiolated seedlings with kinetics similar to those of endogenous rice phytochromes (half-life approximately 20 min). Although plants overexpressing phytochrome A were phenotypically indistinguishable from wild-type plants when grown under high-fluence white light, they were more sensitive as etiolated seedlings to light pulses that established very low phytochrome equilibria. This indicates that the introduced oat phytochrome A was biologically active. Thus, rice ectopically expressing PHY genes may offer a useful model to help understand the physiological functions of the various phytochrome isoforms in monocotyledonous plants.     

Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162–170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.Abbreviations FR far-red light - R red light - WL white light - WL + FR white light supplemented with FR - HIR high-irradiance response - PAR photosynthetically active radiation - Pr, Pfr R- and FR-absorbing forms of phytochrome - Ptot total phytochrome - phyA (PhyA) gene (encoded protein) for phytochrome - WT wild type This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.M.; J.R. Cherry and R.D. Vierstra, (Department of Horticulture, University of Wisconsin-Madison, USA) are thanked for the provision of the transgenic tobacco line.     

Differential usage of photoreceptors for chalcone synthase gene expression during plant development

Photoregulation of chalcone synthase (CHS) mRNA accumulation was analysed in parsley (Petroselinum crispum) and mustard (Sinapis alba) plants at different developmental stages. In both species, CHS mRNA accumulation in young etiolated seedlings was primarily under phytochrome control. In leaves of adult re-etiolated plants, a UV-B photoreceptor was predominantly involved in photocontrol. The reduced red light control in mature leaves was not due to the absence of immunoreactive phytochrome. The apparent dependence of photoreceptor usage on the developmental state of the cell or organism was in accordance with observations on the photoregulation of fusion constructs between CHS promoters from parsley or mustard and the β-glucuronidase reporter gene (GUS). When tested in the parsley protoplast transient expression system, both constructs yielded the same type of photoregulation as observed for the endogenous CHS gene.     

The influence of de-etiolation on the sensitivity of Avena coleoptiles to the far-red absorbing form of phytochrome

In photoresponses regulated by phytochrome the effect of a red irradiation is not always reversed by far-red. This applies for instance to the influence of red light on the geotropic reactions of Avena coleoptiles. We could induce red/far-red reversibility by a short de-etiolating exposure to red light about 20 h prior to the experimental irradiations. This, was due to a decrease of the sensitivity to the low level of the far-red absorbing form of phytochrome that is established by far-red. Since etiolated plants react also to a wavelength of 520 nm (green light), it is advisable to expose the coleoptiles to a de-etiolating irradiation prior to manipulations in green safelight in order to prevent the plants from reacting to the green light.     

Rice Phytochrome Is Biologically Active in Transgenic Tobacco

To investigate the mechanisms of phytochrome action in vivo, we have overexpressed rice phytochrome in transgenic tobacco plants. A full-length rice phytochrome cDNA was fused to the cauliflower mosaic virus 35S promoter and transferred to tobacco. The progeny of some of the transgenic plants contain large amounts of rice phytochrome mRNA in green leaves. Extracts prepared from overexpressing plants contain twofold to fivefold more spectrophotometrically detectable phytochrome than extracts from control plants. Species-specific, anti-phytochrome monoclonal antibodies were used in immunoblots to discriminate between rice and tobacco phytochrome apoproteins in fractions eluted from a DEAE-Sepharose column. Red minus far-red difference spectra of the partially purified rice phytochrome from the transgenic plants indicate that the rice phytochrome assembles with chromophore and is photoreversible. Analysis of the circadian pattern of Cab mRNA levels in transgenic plants versus controls demonstrates that the overproduction of rice phytochrome extends the duration of the free-running rhythm of Cab gene expression. The rice phytochrome is, therefore, biologically active in the transgenic tobacco plant, which establishes a system for in vivo functional analysis of phytochrome.     

The effect of monochromatic red light on ethylene production in leaves of Impatiens balsamina L. and other species

The effect of red and white light on ethylene production was investigated in several plant species. In most cases light inhibited ethylene production. However, stimulation or no effect were also observed in a few species. In those plants where light inhibited ethylene synthesis, the effect of red light was much stronger than that of white light.Both red and white light inhibited ethylene production in green and etiolated seedlings and green leaves of Impatiens balsamina L. The inhibitory effect of red light was stronger than that of white light and much more pronounced when the plants were pretreated with ACC. The effect of red light could be reversed by far-red light. These results suggest that light affects the ethylene forming enzyme (EFE) activity and that its action is mediated by phytochrome.     

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.     

Studies on Greening of Etiolated Seedlings II. Leaf Greening by Phytochrome Action in the Embryonic Axis

We could demonstrate that greening of primary bean leaves in etiolated seedlings of Phaseolus vulgaris cv. Limburg can be controlled by a selective light-pretreatment of the embryonic axis. This light-induced interorgan synergism proved to be a phytochrome-mediated process. The red/farred photoreversible effect on the embryonic axis seems to be primarily linked to changes in the energy metabolism of the primary leaves. Phototransformation of the protochlorophyll present and pigment synthesis are very dependent upon an adequate supply of biochemical energy. When the embryonic axis is selectively pre-exposed to red light for a short time, respiration is markedly enhanced in the leaves and photosynthesis starts immediately upon illumination of the etiolated leaves after an incubation period of optimal length in the dark. The stimulatory effect of the red pretreatment on leaf respiration and photosynthetic capacity could be abolished to the level of the dark controls by a subsequent far-red irradiation on the embryonic axis. It is therefore postulated that phytochrome plays a regulatory role in interorgan cooperation. The metabolic changes involved in photomorphogenesis of etiolated seedlings are closely related to changes in energy production. Our data indicate that the primary act of phytochrome becomes operative at the biochemical level by its directional influence on the energy balance of the cell and coordinates the use of metabolic energy within a tissue and between organs.