Modulation of a mitochondrial function by oat phytochrome in vitro

Previous data in the literature have indicated that phytochrome could alter the rate of reduction of exogenously added NADP by a pea mitochondrial preparation in vitro. These results could not be duplicated using a mitochondrial preparation isolated from etiolated oat seedlings. Further experimentation demonstrated that the addition of Pr to the preparation, in combination with a far red light illumination, could significantly reduce the rate of oxidation of NADH by the external dehydrogenases of oat mitochondria. This response was characterized by a 15% decrease in reaction velocity at saturating substrate concentrations and a 2-fold increase in apparent K_m as compared to values obtained after Pfr plus red light treatment. The response was photoreversible, the rate of oxidation of exogenous NADH being determined by the last light illumination given to the mitochondrial preparation. The interaction between phytochrome and the mitochondria was apparently occurring at the level of the inner mitochondrial membrane. A requirement for these results was that the mitochondria be isolated from plants that were illuminated with white or red light before extraction; mitochondria from unirradiated plants showed no dehydrogenase response to treatments with phytochrome plus actinic light.     

Characterization of the Phytochrome-containing Particles Obtained by Glutaraldehyde Pre-fixation of Maize Coleptiles

Almost all of the particulate phytochrome of maize coleoptiles,obtained by cross-linking phytochrome to its putative receptormembrane by glutaraldehyde within the intact cells, sedimentedduring relatively low-speed centrifugation (10 000-20 000 g).The sediment contained morphologically identifiable mitochondria,some microsomes, plasma membranes, and membranes whose originis not yet known. Although its density was very similar to thatof mitochondria the phytochrome-containing material sedimentedseparately from mitochondria and microsomes but roughly in parallelwith an oligomycin-insensitive ATP-hydrolysing activity. Enrichmentof the phytochrome-containing material was obtained by subfractionationof the 20 000 g sediment. This enrichment of phytochrome coincidedwith an enrichment of certain vesicles, which, on the basisof their staining properties, are taken to be plasma membranevesicles, in the subfraction. The evidence is held to supportthe view that phytochrome attaches to and interacts with theplasma membrane upon photo transformation to the Pfr1 form.     

The anatomy of phytochrome,a unique photoreceptor in plants

Red and far-red light control of plant growth and development is mediated by the photoreceptor phytochrome. The way plants utilize red and far-red light is unique in nature, as are the molecular properties of phytochrome, the molecule that provides the mechanistic basis for this type of light perception. Much of what we know about how plants perceive red lights has come from research on the structure and function of this photoreceptor. This review discusses the main structural features of phytochrome and some new ideas concerning the relationship between phytochrome structure and function. We propose that phytochrome functions as a dimer and that receptor recognition of phytochrome depends on its gross conformation. We also describe a conserved amino acid repeat within the phytochrome molecule and propose that this repeat is important for dimerization and/or phototransformation.     

Destruction and possible de novo synthesis of phytochrome in subcellular fractions of laminae from Avena sativa L.

Phytochrome was determined in etiolated laminae of Avena sativaL. either without pretreatment or after 5 min of red irradiation followed by different periods of darkness (0–24 h). At given intervals laminae were homogenized and phytochrome was determined spectrophotometrically in the total homogenate and in purified etioplasts and mitochondria. Enhanced specific activity of phytochrome was found in all fractions after the irradiation in comparison to dark controls. Phytochrome destruction was observed in all fractions at the beginning of the subsequent dark period. Whereas the homogenate and the mitochondrial fraction showed a continuous destruction so that phytochrome reached a level far below that in etiolated plants, the phytochrome level in the plastid fraction reacheda minimum at 2 h with a subsequent increase beyond the dark level. This increase was most pronounced between 4 and 8 h after the red irradiation. The results are discussed in terms of the destruction and possible de novo synthesis of phytochrome that may be different in mitochondria and plastids.Abbreviations P_tot total phytochrome - P_r red absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - ER endoplasmic reticulum     

In vitro evaluation of mitochondrial–chloroplast subcellular localization of heme oxygenase1 (HO1) in Glycine max

Heme oxygenase1 (HO1) catalyzes the degradation of heme in to biliverdin, carbon monoxide, and ferrous ions. Its role in higher plants has been found as an antioxidant and precursor of phytochrome synthesis. The present study focuses on subcellular localization of HO1 in leaves of soybean has been investigated. Most activity appeared to be located within chloroplast due to its role in phytochrome synthesis but mitochondria also share its localization. Mitochondrial location of HO1 might be on its inner membranous space due to its role in the synthesis of electron donor species which facilitates HO1 catalyzed reaction. Study reports the co-localization of HO1 in both chloroplast and mitochondria.     

Modulation of oat mitochondrial ATPase activity by CA2+ and phytochrome

The activity of a Mg(2+)-dependent ATPase present in highly purified preparations of Avena mitochondria was photoreversibly modulated by red/far-red light treatments. These results were obtained either with mitochondria isolated from plants irradiated with white light prior to the extraction or with mitochondria isolated from unirradiated plants only when purified phytochrome was exogenously added to the reaction mixture. Red light, which converts phytochrome to the far red-absorbing form (Pfr) depressed the ATPase activity, and far-red light reversed this effect. Addition of exogenous CaCl2 also depressed the ATPase activity, and the kinetics of inhibition were similar to the kinetics of the Pfr effects on the ATPase. The calcium chelator, ethyleneglycol-bis(beta-amino-ethyl ether)-N,N' -tetraacetic acid, blocked the effects of both CaCl2 and Pfr on the ATPase. These results are consistent with the interpretation that Pfr promotes a release of Ca2+ from the mitochondrial matrix, thereby inducing an increase in the concentration of intermembranal and extramitochondrial Ca2+.     

Fungal phytochrome chromophore biosynthesis at mitochondria

Mitochondria are essential organelles because of their function in energy conservation. Here, we show an involvement of mitochondria in phytochrome‐dependent light sensing in fungi. Phytochrome photoreceptors are found in plants, bacteria, and fungi and contain a linear, heme‐derived tetrapyrrole as chromophore. Linearization of heme requires heme oxygenases (HOs) which reside inside chloroplasts in planta. Despite the poor degree of conservation of HOs, we identified two candidates in the fungus Alternaria alternata. Deletion of either one phenocopied phytochrome deletion. The two enzymes had a cooperative effect and physically interacted with phytochrome, suggesting metabolon formation. The metabolon was attached to the surface of mitochondria with a C‐terminal anchor (CTA) sequence in HoxA. The CTA was necessary and sufficient for mitochondrial targeting. The affinity of phytochrome apoprotein to HoxA was 57,000‐fold higher than the affinity of the holoprotein, suggesting a “kiss‐and‐go” mechanism for chromophore loading and a function of mitochondria as assembly platforms for functional phytochrome. Hence, two alternative approaches for chromophore biosynthesis and insertion into phytochrome evolved in plants and fungi.     

Association of dehydrins with wheat mitochondria during low-temperature adaptation

In mitochondria from the crowns of field-grown winter wheat plants or their seedlings hardened in the laboratory, thermostable proteins immunologically related to dehydrins were detected. It was found that two dehydrins with mol wts of 63 and 52 kD bound with the outer mitochondrial membrane during autumnal hardening or during adaptation to low temperature in the laboratory. Dehydrins of similar mol wts were detected among proteins in the total membrane fraction from low-temperature-adapted wheat plants. In addition, dehydrins with mol wts of 209 and 196 kD were present in this fraction as well. Dehydrins of similar mol wts were bound with mitochondria from seedlings adapted to low temperature and those from the crowns of plants after autumnal hardening. In spring, the amount of dehydrins associated with mitochondria from the crowns declined to the level characteristic of early autumn. Dehydrin association with mitochondria is evidently an important defense mechanism of frost-resistant plants.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 221–226.Original Russian Text Copyright © 2005 by Borovskii, Stupnikova, Antipina, Anuchina, Voinikov.This revised version was published online in April 2005 with a corrected cover date.     

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.     

Association of Phytochrome with Rough-surfaced Endoplasmic Reticulum Fractions from Soybean Hypocotyls

Distribution of phytochrome (as Pfr) among membranes from soybean hypocotyls (Glycine max L. cv. Wayne) was determined by the combined techniques of cell fractionation, difference spectrometry, and electron microscopic morphometry. More than 90% of the phytochrome was found in the soluble fraction. With homogenates prepared in the presence or absence of Mg²⁺, the portion associated with membrane was only 6.5% and 1%, respectively. In the presence of Mg²⁺, the content of particulate phytochrome correlated with the amount of endoplasmic reticulum with attached ribosomes in the fractions but not with mitochondria or other membranes (including endoplasmic reticulum membranes from which the ribosomes may have been lost during cell fractionation). In the absence of Mg²⁺, phytochrome was associated with a “heavy” plasma membrane fraction. The phytochrome content was sufficiently low to be accounted for by a contamination of less than 10% by rough-surfaced fragments of endoplasmic reticulum. The findings show association of phytochrome with a particulate fraction enriched in rough-surfaced fragments of endoplasmic reticulum but do not rule out cosedimentation of some unknown or unspecific phytochrome aggregate with this fraction.     

Photoresponses of transgenic Arabidopsis overexpressing the fern Adiantum capillus-veneris PHY1.

The phytochrome gene (PHY1) cDNA from the fern Adiantum capillus-veneris encodes an amino acid sequence that shows equal similarity (50-60%) to all five Arabidopsis phytochromes (PHYA-E). The A. capillus-veneris PHY1 cDNA was transformed into Arabidopsis ecotype Landsberg erecta to investigate its activity in angiosperms. Three of the resulting lines contained at least 8 times more spectrally active phytochrome than the wild type, indicating that A. capillus-veneris phytochrome can incorporate the chromophore of the host plants. Hypocotyl growth inhibition of these transgenic lines was investigated under red and far-red light. The results indicated dominant negative activity of A. capillus-veneris phy1 on the phytochrome A response in the host plants under continuous far-red light. However, the fern phytochrome did not interfere with the red-light repression of hypocotyl growth mediated by endogenous phytochrome B, and it failed to complement a phyB mutant phenotype. These observations suggest that the phy1 phytochrome molecule is too diverged from those of Arabidopsis to be fully functional.     

高等植物光敏色素的分子结构、生理功能和进化特征

光敏色素是植物感受外界环境变化的最重要光受体之一, 对红光和远红外光非常敏感。本文综述了光敏色素的分子结构、它所包含的结构域和相应功能以及植物各主要类群中光敏色素基因家族的成员组成与进化关系; 重点在分子水平上介绍了光敏色素的生理功能与作用机制。最后, 基于最新的研究进展提出了将来的研究方向。     

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.     

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.     

Light-independent phytochrome signaling mediated by dominant GAF domain tyrosine mutants of Arabidopsis phytochromes in transgenic plants

The photoreversibility of plant phytochromes enables continuous surveillance of the ambient light environment. Through expression of profluorescent, photoinsensitive Tyr-to-His mutant alleles of Arabidopsis thaliana phytochrome B (PHYB(Y276H)) and Arabidopsis phytochrome A (PHYA(Y242H)) in transgenic Arabidopsis plants, we demonstrate that photoconversion is not a prerequisite for phytochrome signaling. PHYB(Y276H)-expressing plants exhibit chromophore-dependent constitutive photomorphogenesis, light-independent phyB(Y276H) nuclear localization, constitutive activation of genes normally repressed in darkness, and light-insensitive seed germination. Fluence rate analyses of transgenic plants expressing PHYB(Y276H), PHYA(Y242H), and other Y(GAF) mutant alleles of PHYB demonstrate that a range of altered light-signaling activities are associated with mutation of this residue. We conclude that the universally conserved GAF domain Tyr residue, with which the bilin chromophore is intimately associated, performs a critical role in coupling light perception to signal transduction by plant phytochromes.     

Phytochrome-mediated control of respiratory activities of mitochondria in cotyledons of dark-grown cucumber seedlings

Mitochondria isolated from cotyledons of dark-grown cucumber ( Cucumber sativus L., cv. Shimotsuki-Aonaga) seedlings after illumination with continuous far-red light showed an increased capacity for oxidation of malate or α-ketoglutarate, as compared with those from cotyledons of non-illuminated seedlings. This increase is supposed to be caused by phytochrome action (high irradiance response). Exogenous NAD⁺ had no effect on the rate of the oxidation of α-ketoglutarate or malate by mitochondria isolated from far-red light-treated cotyledons, but it enhanced the oxidation rate of mitochondria from control cotyledons to the level of mitochondria from light-treated ones. The NAD (NAD⁺+ NADH) content was higher in mitochondria isolated from continuously far-red light-treated cotyledons than in mitochondria from controls. The NAD content was also increased by the treatment with a red light pulse and this response was reversed by a subsequent far-red light pulse. It is proposed that phytochrome controls respiratory activities of cucumber mitochondria by changing the size of the NAD pool in the mitochondria.     

The Phytochrome-Deficient pcd1 Mutant of Pea Is Unable to Convert Heme to Biliverdin IX[alpha

We isolated a new pea mutant that was selected on the basis of pale color and elongated internodes in a screen under white light. The mutant was designated pcd1 for phytochrome chromophore deficient. Light-grown pcd1 plants have yellow-green foliage with a reduced chlorophyll (Chl) content and an abnormally high Chl a/Chl b ratio. Etiolated pcd1 seedlings are developmentally insensitive to far-red light, show a reduced response to red light, and have no spectrophotometrically detectable phytochrome. The phytochrome A apoprotein is present at the wild-type level in etiolated pcd1 seedlings but is not depleted by red light treatment. Crude phytochrome preparations from etiolated pcd1 tissue also lack spectral activity but can be assembled with phycocyanobilin, an analog of the endogenous phytochrome chromophore phytochromobilin, to yield a difference spectrum characteristic of an apophytochrome-phycocyanobilin adduct. These results indicate that the pcd1-conferred phenotype results from a deficiency in phytochrome chromophore synthesis. Furthermore, etioplast preparations from pcd1 seedlings can metabolize biliverdin (BV) IX[alpha] but not heme to phytochromobilin, indicating that pcd1 plants are severely impaired in their ability to convert heme to BV IX[alpha]. This provides clear evidence that the conversion of heme to BV IX[alpha] is an enzymatic process in higher plants and that it is required for synthesis of the phytochrome chromophore and hence for normal photomorphogenesis.     

Intramitochondrial transfer of phospholipids in the yeast, Saccharomyces cerevisiae

Translocation of phosphatidylinositol, which is synthesized on the outer aspect of the outer membrane of isolated yeast mitochondria, to the inner membrane is linked to phosphatidylinositol synthesis and is therefore a vectorial process. Phosphatidylinositol once integrated into the inner mitochondrial membrane is not transferred back to the mitochondrial surface. Phosphatidylserine is also translocated from the outer to the inner mitochondrial membrane, where it is decarboxylated to phosphatidylethanolamine. We made use of this metabolic modification to characterize the intramitochondrial transfer of phosphatidylserine and phosphatidylethanolamine. Intramitochondrial phosphatidylserine transfer is insensitive to the uncoupler carbonyl cyanide m-chlorophenylhydrazone and to valinomycin and is thus independent of an electrochemical gradient across the inner membrane. Transfer of phosphatidylserine from the outer to the inner mitochondrial membrane occurs not only in intact mitochondria but also in mitoplasts which are devoid of intermembrane space proteins but have the outer membrane still adherent to the inner membrane. This result suggests that specific contact sites are involved in the intramitochondrial translocation of phospholipids. 3H-Labeled phosphatidylethanolamine synthesized from [3H]serine in isolated mitochondria is readily exported from the inner to the outer mitochondrial membrane without prior mixing with the pool of phosphatidylethanolamine of the inner membrane.