Phototropin-related NPL1 controls chloroplast relocation induced by blue light.

In photosynthetic cells, chloroplasts migrate towards illuminated sites to optimize photosynthesis and move away from excessively illuminated areas to protect the photosynthetic machinery. Although this movement of chloroplasts in response to light has been known for over a century, the photoreceptor mediating this process has not been identified. The Arabidopsis gene NPL1 (ref. 2) is a paralogue of the NPH1 gene, which encodes phototropin, a photoreceptor for phototropic bending. Here we show that NPL1 is required for chloroplast relocation induced by blue light. A loss-of-function npl1 mutant showed no chloroplast avoidance response in strong blue light, whereas the accumulation of chloroplasts in weak light was normal. These results indicate that NPL1 may function as a photoreceptor mediating chloroplast relocation.

An Arabidopsis circadian clock component interacts with both CRY1 and phyB.

Most organisms, from cyanobacteria to mammals, use circadian clocks to coordinate their activities with the natural 24-h light/dark cycle. The clock proteins of Drosophila and mammals exhibit striking homology but do not show similarity with clock proteins found so far from either cyanobacteria or Neurospora. Each of these organisms uses a transcriptionally regulated negative feedback loop in which the messenger RNA levels of the clock components cycle over a 24-h period. Proteins containing PAS domains are invariably found in at least one component of the characterized eukaryotic clocks. Here we describe ADAGIO1 (ADO1), a gene of Arabidopsis thaliana that encodes a protein containing a PAS domain. We found that a loss-of-function ado1 mutant is altered in both gene expression and cotyledon movement in circadian rhythmicity. Under constant white or blue light, the ado1 mutant exhibits a longer period than that of wild-type Arabidopsis seedlings, whereas under red light cotyledon movement and stem elongation are arrhythmic. Both yeast two-hybrid and in vitro binding studies show that there is a physical interaction between ADO1 and the photoreceptors CRY1 and phyB. We propose that ADO1 is an important component of the Arabidopsis circadian system.

Cryptochromes: blue light receptors for plants and animals.

Cryptochromes are blue, ultraviolet-A photoreceptors. They were first characterized for Arabidopsis and are also found in ferns and algae; they appear to be ubiquitous in the plant kingdom. They are flavoproteins similar in sequence to photolyases, their presumptive evolutionary ancestors. Cryptochromes mediate a variety of light responses, including entrainment of circadian rhythms in Arabidopsis, Drosophila, and mammals. Sequence comparison indicates that the plant and animal cryptochrome families have distinct evolutionary histories, with the plant cryptochromes being of ancient evolutionary origin and the animal cryptochromes having evolved relatively recently. This process of repeated evolution may have coincided with the origin in animals of a modified circadian clock based on the PERIOD, TIMELESS, CLOCK, and CYCLE proteins.

Low temperature regulates Arabidopsis Lhcb gene expression in a light-independent manner.

Low temperature treatment of dark-grown seedlings of Arabidopsis thaliana results in a rapid increase in the amount of mRNAs encoding for the major polypeptides of the light-harvesting complex of photosystem II (Lhcb1 genes). This increase is transient and seems to be due mainly to the accumulation of Lhcb1*3 transcripts, indicating that low temperature differentially regulates the expression of the Arabidopsis Lhcb1 gene family in the dark. A 1.34 kb fragment of the Lhcb1*3 promoter is sufficient to confer low temperature regulation to a reporter gene in transgenic Arabidopsis etiolated seedlings, suggesting that the regulation is occurring at the transcriptional level. The cold-induced accumulation of Lhcb1*3 mRNA is not part of a general response to stressful conditions since no accumulation is detected in response to water stress, anaerobiosis or salt stress. The amount of Lhcb1*3 mRNA decrease in response to exogenous abscisic acid (ABA) suggesting that this phytohormone acts as a negative regulator. Moreover, the accumulation of Lhcb1*3 mRNAs in cold-treated ABA deficient etiolated seedlings is higher than that of wild-type and ABA insensitive etiolated seedlings, indicating that low temperature regulation of Lhcb1*3 is not mediated by ABA.

The CRY1 blue light photoreceptor of Arabidopsis interacts with phytochrome A in vitro.

Plants have at least two major photosensory receptors: phytochrome (absorbing primarily red/far-red light) and cryptochrome (absorbing blue/UV-A light); considerable physiological and genetic evidence suggests some form of communication or functional dependence between the receptors. Here, we demonstrate in vitro, using purified recombinant photoreceptors, that Arabidopsis CRY1 and CRY2 (cryptochrome) are substrates for phosphorylation by a phytochrome A-associated kinase activity. Several mutations within the CRY1 C terminus lead to reduced phosphorylation by phytochrome preparations in vitro. Yeast two-hybrid interaction studies using expressed C-terminal fragments of CRY1 and phytochrome A from Arabidopsis confirm a direct physical interaction between both photoreceptors. In vivo labeling studies and specific mutant alleles of CRY1, which interfere with the function of phytochrome, suggest the possible relevance of these findings in vivo.

Cryptochrome blue-light photoreceptors of Arabidopsis implicated in phototropism.

Phototropism-bending towards the light-is one of the best known plant tropic responses. Despite being reported by Darwin and others over a century ago to be specifically under the control of blue light, the photoreceptors mediating phototropism have remained unknown. We have characterized a blue-light photoreceptor from Arabidopsis, named CRY1 for cryptochrome 1; this photoreceptor is a flavoprotein that mediates numerous blue-light-dependent responses. In Arabidopsis, HY4 (the gene encoding CRY1) is a member of a small gene family that also encodes a related photoreceptor, CRY2, which shares considerable functional overlap with CRY1. Here we report that mutant plants lacking both the CRY1 and the CRY2 blue-light photoreceptors are deficient in the phototropic response. Transgenic Arabidopsis plants overexpressing CRY1 or CRY2 show enhanced phototropic curvature. We conclude that cryptochrome is one of the photoreceptors mediating phototropism in plants.

Chimeric proteins between cry1 and cry2 Arabidopsis blue light photoreceptors indicate overlapping functions and varying protein stability.

A blue light (cryptochrome) photoreceptor from Arabidopsis, cry1, has been identified recently and shown to mediate a number of blue light-dependent phenotypes. Similar to phytochrome, the cryptochrome photoreceptors are encoded by a gene family of homologous members with considerable amino acid sequence similarity within the N-terminal chromophore binding domain. The two members of the Arabidopsis cryptochrome gene family (CRY1 and CRY2) overlap in function, but their proteins differ in stability: cry2 is rapidly degraded under light fluences (green, blue, and UV) that activate the photoreceptor, but cry1 is not. Here, we demonstrate by overexpression in transgenic plants of cry1 and cry2 fusion constructs that their domains are functionally interchangeable. Hybrid receptor proteins mediate functions similar to cry1 and include inhibition of hypocotyl elongation and blue light-dependent anthocyanin accumulation; differences in activity appear to be correlated with differing protein stability. Because cry2 accumulates to high levels under low-light intensities, it may have greater significance in wild-type plants under conditions when light is limited.

Two homologous low-temperature-inducible genes from Arabidopsis encode highly hydrophobic proteins.

We have characterized two related cDNAs (RCI2A and RCI2B) corresponding to genes from Arabidopsis thaliana, the expression of which is transiently induced by low, nonfreezing temperatures. RCI2A and RCI2B encode small (54 amino acids), highly hydrophobic proteins that bear two potential transmembrane domains. They show similarity to proteins encoded by genes from barley (Hordeum vulgare L.) and wheatgrass (Lophophyrum elongatum) that are regulated by different stress conditions. Their high level of sequence homology (78%) and their genomic location in a single restriction fragment suggest that both genes originated as a result of a tandem duplication. However, their regulatory sequences have diverged enough to confer on them different expression patterns. Like most of the cold-inducible plant genes characterized, the expression of RCI2A and RCI2B is also promoted by abscisic acid (ABA) and dehydration but is not a general response to stress conditions, since it is not induced by salt stress or by anaerobiosis. Furthermore, low temperatures are able to induce RCI2A and RCI2B expression in ABA-deficient and -insensitive genetic backgrounds, indicating that both ABA-dependent and -independent pathways regulate the low-temperature responsiveness of these two genes.

Species-specific voltage-gating properties of connexin-45 junctions expressed in Xenopus oocytes.

Gap junctions composed of connexin-45 (Cx45) homologs from four species, zebrafish, chicken, mouse, and human, were expressed in pairs of Xenopus oocytes. The macroscopic conductance (gj) of all Cx45 junctions was modulated by transjunctional voltage (Vj) and by the inside-outside voltage (Vm), and the modulation was species specific. Although their gating characteristics varied in voltage sensitivity and kinetics, the four Cx45 junctions shared 1) maximum conductance at Vj = 0 and symmetrical gj reduction in response to positive and negative Vj of low amplitude, with little residual conductance; and 2) gj increases in response to simultaneous depolarization of the paired cells. The formation of hybrid channels, comprising Cx45 hemichannels from different species, allowed us to infer that two separate gates exist, one in each hemichannel, and that each Cx45 hemichannel is closed by the negativity of Vj on its cytoplasmic side. Interestingly, the Vm dependence of hybrid channels also suggests the presence of two gates in series, one Vm gate in each hemichannel. Thus the Vj and Vm dependence provides evidence that two independent voltage gates in each Cx45 hemichannel exist, reacting through specific voltage sensors and operating by different mechanisms, properties that have evolved divergently among species.

An enzyme similar to animal type II photolyases mediates photoreactivation in Arabidopsis.

The important issue of photoreactivation DNA repair in plants has become even more interesting in recent years because a family of genes that are highly homologous to photoreactivating DNA repair enzymes but that function as blue light photoreceptors has been isolated. Here, we report the isolation of a novel photolyase-like sequence from Arabidopsis designated PHR1 (for photoreactivating enzyme). It shares little sequence similarity with either type I photolyases or the cryptochrome family of blue light photoreceptors. Instead, the PHR1 gene encodes an amino acid sequence with significant homology to the recently characterized type II photolyases identified in a number of prokaryotic and animal systems. PHR1 is a single-copy gene and is not expressed in dark-grown etiolated seedlings: the message is light inducible, which is similar to the expression profile for photoreactivation activity in plants. The PHR1 protein complements a photolyase-deficient mutant of Escherichia coli and thus confers photoreactivation activity. In addition, an Arabidopsis mutant that is entirely lacking in photolyase activity has been found to contain a lesion within this Arabidopsis type II photolyase sequence. We conclude that PHR1 represents a genuine plant photolyase gene and that the plant genes with homology to type I photolyases (the cryptochrome family of blue light photoreceptors) do not contribute to photoreactivation repair, at least in the case of Arabidopsis.

Low Temperature Induces the Accumulation of Phenylalanine Ammonia-Lyase and Chalcone Synthase mRNAs of Arabidopsis thaliana in a Light-Dependent Manner.

Anthocyanins, which accumulate in leaves and stems in response to low temperature and changes in light intensity, are synthesized through the phenylpropanoid pathway that is controlled by key enzymes that include phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS). In this work we demonstrate that PAL and CHS mRNAs accumulate in leaves of Arabidopsis thaliana (L.) Heynh. upon exposure to low temperature in a light-dependent manner. The regulation of the PAL1 gene expression by low temperature and light was examined by analyzing the expression of the [beta]-glucuronidase (uidA) reporter gene in transgenic Arabidopsis plants containing the uidA gene of Escherichia coli under the control of the PAL1 promoter. The results indicate that the accumulation of PAL1 mRNA is transcriptionally regulated. Histochemical staining for [beta]-glucuronidase activity showed that the PAL1 promoter is preferentially activated in photosynthetically active cells, paralleling anthocyanin accumulation. Moreover, we show that light may also be implicated in the regulation of the CHS gene in response to bacterial infiltration. Finally, using two transparent testa Arabidopsis mutants that are unable to accumulate anthocyanins, we demonstrate that these pigments are not required for successful development of freezing tolerance in this species.

Bovine connexin44, a lens gap junction protein: molecular cloning, immunologic characterization, and functional expression.

PURPOSE: To identify, clone molecularly, characterize immunochemically, and express functionally a bovine lens gap junction protein (connexin). METHODS: The methods used were polymerase chain reaction, genomic cloning, RNA and DNA blotting, bacterial expression of a fusion protein, immunoblotting, alkaline phosphatase treatment, Xenopus oocyte expression, and voltage clamp technique. RESULTS: A bovine genomic clone encoding a polypeptide of 44,424 d, termed connexin44 (Cx44), was isolated. Cx44 was most closely related to the lens connexins rat Cx46 and chicken Cx56. The Cx44 DNA hybridized to a 2.5 kb mRNA detected only in lens RNA. The carboxyl terminal 161 amino acids from Cx44 were expressed in bacteria fused to maltose binding protein (MBP). The Cx44/MBP fusion protein reacted in immunoblots with anti-rat Cx46(411 to 416) antibodies and with the monoclonal antibody 5H1, but not with a monoclonal antibody to MP70 nor with antibodies to other connexins. Cx44 translated in vitro from the cloned DNA showed a single band with an apparent electrophoretic mobility of approximately 50 kd on polyacrylamide gels containing sodium dodecyl sulfate. Multiple bands of 53 to 57 kd were detected by immunoblotting in homogenates of bovine lens; these bands were reduced to a broad band of approximately 50 kd by alkaline phosphatase treatment, suggesting that they represented phosphorylated forms of Cx44. Cx44 RNA injected in single oocytes induced a large and characteristic time- and voltage-dependent current. Overexpression of Cx44 produced depolarization and cell lysis. Junctional currents that could be regulated by transjunctional voltage were induced between paired oocytes injected with Cx44 RNA. Observations in paired oocytes suggested the assembly of hemichannels into junctional channels. CONCLUSIONS: Cx44 is a phosphoprotein component of bovine lens fiber gap junctions. Although it has a relatively distinct sequence, it shares sequence similarity, immunologic cross-reactivity, and electrophysiological properties with rat Cx46. These data suggest that Cx44 is the protein previously identified in several immunohistochemical studies of bovine lens gap junctions that used anti-rat Cx46 antibodies. They also suggest that the formation of intercellular channels by pairing of hemichannels might prevent the cell lysis induced by the opening of unpaired hemichannels.

Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins, a family of putative kinase regulators.

We have isolated two Rare Cold-Inducible (RCI1 and RCI2) cDNAs by screening a cDNA library prepared from cold-acclimated etiolated seedlings of Arabidopsis thaliana with a subtracted probe. RNA-blot hybridizations revealed that the expression of both RCI1 and RCI2 genes is induced by low temperature independently of the plant organ or the developmental stage considered. However, RCI1 mRNA accumulates faster and at higher levels than the RCI2 one indicating that these genes have differential responsiveness to cold stress. Additionally, when plants are returned to room temperature, RCI1 mRNA decreases faster than RCI2. In contrast to most of the cold-inducible plant genes characterized, the expression of RCI1 and RCI2 is not induced by ABA or water stress. The nucleotide sequences of RCI1 and RCI2 cDNAs predict two acidic polypeptides of 255 and 251 amino acids with molecular weights of 29 and 28 kDa respectively. The alignment of these polypeptides indicates that they have 181 identical amino acids suggesting that the corresponding genes have a common origin. Sequence comparisons reveal no similarities between the RCI proteins and any other cold-regulated plant protein so far described. Instead, they demonstrate that the RCI proteins are highly homologous to a family of proteins, known as 14-3-3 proteins, which are thought to be involved in the regulation of multifunctional protein kinases.

Low Temperature Induces the Accumulation of Alcohol Dehydrogenase mRNA in Arabidopsis thaliana, a Chilling-Tolerant Plant.

mRNA encoding alcohol dehydrogenase (ADH) increases in etiolated seedlings and leaves of Arabidopsis thaliana (L.) Heynh. upon exposure to low temperature. The analysis of this response after water stress and abscisic acid (ABA) treatments in Arabidopsis wild type and ABA-deficient and -insensitive mutants indicates that cold accumulation of ADH mRNA could be induced by both anaerobic metabolism and increase of ABA concentration resulting from low temperature exposure. By using one Arabidopsis ADH null mutant, we show that ADH activity is not required for successful development of freezing tolerance in this species.