Phytochrome A-specific signaling in Arabidopsis thaliana

Among the five phytochromes in Arabidopsis thaliana, phytochrome A (phyA) plays a major role in seedling de-etiolation. Until now more then ten positive and some negative components acting downstream of phyA have been identified. However, their site of action and hierarchical relationships are not completely understood yet.     

Roles of different phytochromes in Arabidopsis photomorphogenesis

The red/far-red light-absorbing phytochromes play fundamental roles in photoperception of the light environment and the subsequent adaptation of plant growth and development. Higher plants possess multiple, discrete phytochromes, the apoproteins of which are the products of a family of divergent (PHY) genes. Arabidopsis thaliana has at least five PHY genes, encoding the apoproteins of phytochromes A-E. Through the analysis of mutants that are deficient in phytochrome A or B and the corresponding double mutant, it is becoming clear that these phytochromes perform both discrete and overlapping roles throughout plant development. Through analysis of the phyA phyB double mutant, it has been possible to define several responses that are mediated by other members of the phytochrome family. This article reviews some of the recent progress in the study of phytochrome-deficient mutants of the model plant Arabidopsis thaliana.     

The Histidine Kinase-Related Domain of Arabidopsis Phytochrome A Controls the Spectral Sensitivity and the Subcellular Distribution of the Photoreceptor

Phytochrome A (phyA) is the primary photoreceptor for sensing extremely low amounts of light and for mediating various far-red light-induced responses in higher plants. Translocation from the cytosol to the nucleus is an essential step in phyA signal transduction. EID1 (for EMPFINDLICHER IM DUNKELROTEN LICHT1) is an F-box protein that functions as a negative regulator in far-red light signaling downstream of the phyA in Arabidopsis (Arabidopsis thaliana). To identify factors involved in EID1-dependent light signal transduction, pools of ethylmethylsulfonate-treated eid1-3 seeds were screened for seedlings that suppress the hypersensitive phenotype of the mutant. The phenotype of the suppressor mutant presented here is caused by a missense mutation in the PHYA gene that leads to an amino acid transition in its histidine kinase-related domain. The novel phyA-402 allele alters the spectral sensitivity and the persistence of far-red light-induced high-irradiance responses. The strong eid1-3 suppressor phenotype of phyA-402 contrasts with the moderate phenotype observed when phyA-402 is introgressed into the wild-type background, which indicates that the mutation mainly alters functions in an EID1-dependent signaling cascade. The mutation specifically inhibits nuclear accumulation of the photoreceptor molecule upon red light irradiation, even though it still interacts with FHY1 (for far-red long hypocotyl 1) and FHL (for FHY1-like protein), two factors that are essential for nuclear accumulation of phyA. Degradation of the mutated phyA is unaltered even under light conditions that inhibit its nuclear accumulation, indicating that phyA degradation may occur mostly in the cytoplasm.     

Light regulation of succinate dehydrogenase expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves

A potential mechanism of light regulation of the succinate dehydrogenase (SDH) expression in Arabidopsis thaliana leaves was studied. As was shown by dot-hybridization and polymerase chain reaction in real time (RT-PCR), the SDH mRNA level in wild-type Arabidopsis thaliana plants changed depending on light conditions. The level of SDH mRNA in darkness was higher than in the light. The analysis of Arabidopsis thaliana plants carrying the mutant genes of phytochromes A and B showed that phytochrome A was involved in the regulation of the SDH enzyme activity. The active form of phytochrome A suppressed the SDHI-2 gene expression, and that resulted in decreasing activity of SDH.     

PNZIP Is a Novel Mesophyll-Specific cDNA That Is Regulated by Phytochrome and a Circadian Rhythm and Encodes a Protein with a Leucine Zipper Motif

Single, double, and triple null combinations of Arabidopsis mutants lacking the photoreceptors phytochrome (phy) A (phyA-201), phyB (phyB-5), and cryptochrome (cry) 1 (hy4-2.23n) were examined for de-etiolation responses in high-fluence red, far-red, blue, and broad-spectrum white light. Cotyledon unhooking, unfolding, and expansion, hypocotyl growth, and the accumulation of chlorophylls and anthocyanin in 5-d-old seedlings were measured under each light condition and in the dark. phyA was the major photoreceptor/effector for most far-red-light responses, although phyB and cry1 modulated anthocyanin accumulation in a phyA-dependent manner. phyB was the major photoreceptor in red light, although cry1 acted as a phyA/phyB-dependent modulator of chlorophyll accumulation under these conditions. All three photoreceptors contributed to most blue light deetiolation responses, either redundantly or additively; however, phyB acted as a modulator of cotyledon expansion dependent on the presence of cry1. As reported previously, flowering time in long days was promoted by phyA and inhibited by phyB, with each suppressing the other's effect. In addition to the effector/modulator relationships described above, measurements of hypocotyls from blue-light-grown seedlings demonstrated phytochrome activity in blue light and cry1 activity in a phyAphyB mutant background.     

The rosette habit of Arabidopsis thaliana is dependent upon phytochrome action: novel phytochromes control internode elongation and flowering time

A major function of phytochromes in light-grown plants involves the perception of changes in the relative amounts of red and far-red light (R:FR ratio) and the initiation of the shade-avoidance response. In Arabidopsis thaliana, this response is typified by increased elongation growth of petioles and accelerated flowering and can be fully induced by end-of-day far-red light (EOD FR) treatments. Phytochrome B-deficient (phyB) mutants, which have a constitutive elongated-petiole and early-flowering phenotype, do not display a petiole elongation growth response to EOD FR, but they do respond to EOD FR by earlier flowering. Seedlings deficient in both phytochrome A and phytochrome B (phyA phyB), have a greatly reduced stature compared with wild-type or either monogenic mutant. The phyA phyB double null mutants also respond to EOD FR treatments by flowering early, suggesting the operation of novel phytochromes. Contrary to the behaviour of wild-type or monogenic phyA or phyB seedlings, petiole elongation in phyA phyB seedlings is reduced in response to EOD FR treatments. This reduction in petiole elongation is accompanied by the appearance of elongated internodes such that under these conditions the plants no longer display a rosette habit.     

Not all anthocyanins are born equal: distinct patterns induced by stress in Arabidopsis

Main Conclusion

Different abiotic stress conditions induce distinct sets of anthocyanins, indicating that anthocyanins have different biological functions, or that decoration patterns of each anthocyanin are used for unique purposes during stress. The induction of anthocyanin accumulation in vegetative tissues is often considered to be a response of plants to biotic or abiotic stress conditions. Arabidopsis thaliana (Arabidopsis) accumulates over 20 anthocyanins derived from the anthocyanidin cyanidin in an organ-specific manner during development, but the anthocyanin chemical diversity for their alleged stress protective functions remains unclear. We show here that, when grown in various abiotic stress conditions, Arabidopsis not only often accumulates significantly higher levels of total anthocyanins, but different stress conditions also favor the accumulation of different sets of anthocyanins. For example, the anthocyanin patterns of seedlings grown at pH 3.3 or in media lacking phosphate are very similar and characterized by relatively high levels of the anthocyanins A8 and A11. In contrast, anthocyanin inductive conditions (AIC) provided by high sucrose media are characterized by high accumulation of A9* and A5 relative to other stress conditions. The modifications present in each condition correlate reasonably well with the induction of the respective anthocyanin modification enzymes. Taken together, our results suggest that Arabidopsis anthocyanin profiles provide ‘fingerprints’ that reflect the stress status of the plants.     

Functional analysis of yeast-derived phytochrome A and B phycocyanobilin adducts

Investigations of phytochrome mutants of Arabidopsis suggested that the expression of chalcone synthase ( chs ) and anthocyanin accumulation is predominantly controlled by phytochrome A. To test the functionality of phytochrome A and B at the molecular level recombinant, yeast-derived phytochrome-phycocyanobilin adducts (phyA*, phyB*) and oat phytochrome A (phyA) were microinjected into etiolated aurea tomato seedlings. Subsequent to microinjection anthocyanin and chlorophyll accumulation was monitored as well as β-glucuronidase (GUS) expression mediated by light-regulated promoters ( chs , chlorophyll a/b binding protein ( lhcb1 ) and ferredoxin NADP+ oxidoreductase ( fnr )). Microinjection of phyA* under white light conditions caused anthocyanin and chlorophyll accumulation and mediated chs —GUS, lhcb1 —GUS and fnr —GUS expression. Microinjection of phyB* under identical conditions induced chlorophyll accumulation and mediated lhcb1 —GUS and fnr —GUS expression but neither anthocyanin accumulation nor chs —GUS expression were observed. The characterization of Arabidopsis phytochrome mutants and the microinjection experiments suggested that phyB cannot induce the accumulation of juvenile anthocyanin. Microinjections under far-red light conditions demonstrated that phyA can act independently of other photoreceptors. By contrast, phyB* injections under red light conditions indicated that phyB* needs interactions with other photoreceptors to mediate a rapid and efficient de-etiolation signal.     

Detection of spatial-specific phytochrome responses using targeted expression of biliverdin reductase in Arabidopsis

To regulate levels of holophytochrome in a spatial-specific manner and investigate the major sites of action of phytochromes during seedling development, we constructed transgenic Arabidopsis (Arabidopsis thaliana) plant lines expressing plastid-targeted mammalian biliverdin IXα reductase (pBVR) under regulatory control of CAB3 and MERI5 promoters. Comparative photobiological and phenotypic analyses indicated that spatial-specific expression of pBVR led to the disruption of distinct subsets of phytochrome-regulated responses for different promoters. pBVR expression in photosynthetic tissues (CAB3pBVR lines) had intermediate effects on chlorophyll accumulation, carotenoid production, anthocyanin synthesis, and leaf development responses in white-light conditions. CAB3pBVR expression, however, resulted in distinctive phenotypes in far-red (FR) conditions. A number of FR high irradiance responses were disrupted in CABpBVR lines, including FR-dependent inhibition of hypocotyl elongation and stimulation of anthocyanin accumulation. By contrast, preferential expression of pBVR in the shoot apical meristem in MERI5pBVR lines resulted in a phytochrome-deficient, leaf development phenotype under short-day growth conditions. These results implicate leaf-localized phytochrome A as having a unique role in regulating FR-mediated hypocotyl elongation and meristem- and/or leaf primordia-localized phytochromes as having a novel role in phytochrome-dependent responses. Taken together, these studies demonstrate the efficacy of selectively inactivating distinct phytochrome-mediated responses by regulated expression of BVR in transgenic plants, a novel means to investigate the sites of phytochrome photoperception and to regulate specifically light-mediated plant growth and development.     

Downstream effectors of light- and phytochrome-dependent regulation of hypocotyl elongation in Arabidopsis thaliana

Arabidopsis, like most plants, exhibits tissue-specific, light-dependent growth responses. Cotyledon and leaf growth and the accumulation of photosynthetic pigments are promoted by light, whereas hypocotyl growth is inhibited. The identification and characterization of distinct phytochrome-dependent molecular effectors that are associated with these divergent tissue-specific, light-dependent growth responses are limited. To identify phytochrome-dependent factors that impact the photoregulation of hypocotyl length, we conducted comparative gene expression studies using Arabidopsis lines exhibiting distinct patterns of phytochrome chromophore inactivation and associated disparate hypocotyl elongation responses under far-red (FR) light. A large number of genes was misregulated in plants lacking mesophyll-specific phytochromes relative to constitutively-deficient phytochrome lines. We identified and characterized genes whose expression is impacted by light and by phyA and phyB that have roles in the photoregulation of hypocotyl length. We characterized the functions of several identified target genes by phenotyping of T-DNA mutants. Among these genes is a previously uncharacterized LHE (LIGHT-INDUCED HYPOCOTYL ELONGATION) gene, which we show impacts light- and phytochrome-mediated regulation of hypocotyl elongation under red (R) and FR illumination. We describe a new approach for identifying genes involved in light- and phytochrome-dependent, tissue-specific growth regulation and confirmed the roles of three such genes in the phytochrome-dependent photoregulation of hypocotyl length.     

Nuclear accumulation of the phytochrome A photoreceptor requires FHY1

The phytochrome family of red/far-red (R/FR)-responsive photoreceptors plays a key role throughout the life cycle of plants . Arabidopsis has five phytochromes, phyA-phyE, among which phyA and phyB play the most predominant functions . Light-regulated nuclear accumulation of the phytochromes is an important regulatory step of this pathway, but to this date no factor specifically required for this event has been identified . Among all phyA signaling mutants, fhy1 and fhy3 (far-red elongated hypocotyl 1 and 3) have the most severe hyposensitive phenotype, indicating that they play particularly important roles . FHY1 is a small plant-specific protein of unknown function localized both in the nucleus and the cytoplasm . Here we show that FHY1 is specifically required for the light-regulated nuclear accumulation of phyA but not phyB. Moreover, phyA accumulation is only slightly affected in fhy3, indicating that the diminished nuclear accumulation of phyA observed in fhy1 seedlings is not simply a general consequence of reduced phyA signaling. By in vitro pull-down and yeast two-hybrid analyses, we demonstrate that FHY1 physically interacts with phyA, preferentially in its active Pfr form. Furthermore, FHY1 and phyA colocalize in planta. We therefore identify the first component required for light-regulated phytochrome nuclear accumulation.     

FHY1 and FHL act together to mediate nuclear accumulation of the phytochrome A photoreceptor

The phytochrome family of red/far-red photoreceptors is involved in the regulation of a wide range of developmental responses in plants. The Arabidopsis genome contains five phytochromes (phyA-E), among which phyA and phyB play the most important roles. Phytochromes localize to the cytosol in the dark and accumulate in the nucleus under light conditions, inducing specific phytochrome-mediated responses. Light-regulated nuclear accumulation of the phytochrome photoreceptors is therefore considered a key regulatory step of these pathways. In fact, one of the most severe phyA signaling mutants, fhy1 (far red elongated hypocotyl 1), is strongly affected in nuclear accumulation of phyA. The fhy1 fhl (fhy1 like) double mutant, lacking both FHY1 and its only close homolog FHL, is virtually blind to far-red light like phyA null seedlings. Here we show that FHL accounts for residual amounts of phyA in the nucleus in a fhy1 background and that nuclear accumulation of phyA is completely inhibited in an fhy1 FHL RNAi knock-down line. Moreover, we demonstrate that FHL and phyA interact with each other in a light-dependent manner and that they co-localize in light-induced nuclear speckles. We also identify a phyA-binding site at the C-terminus of FHY1 and FHL, and show that the N-terminal 406 amino acids of phyA are sufficient for the interaction with FHY1/FHL.     

Dynamic properties of endogenous phytochrome A in Arabidopsis seedlings.

The dynamic behavior of phytochrome A (phyA) in seedlings of the model plant Arabidopsis was examined by in vivo spectroscopy and by western and northern blotting. Rapid accumulation of phyA was observed, reaching a steady state after 3 d. Both red and far-red light initiated a rapid destruction of the far-red-light-absorbing form of phytochrome (Pfr); the apparent half-life was only 4-fold longer in far-red than in red light. Furthermore, the Pfr-induced destruction of the red-light-absorbing form of phytochrome (Pr) of phyA occurred in darkness with a rate identical to that of Pfr destruction. A 2-fold decrease in mRNA abundance was observed after irradiation, irrespective of the applied light quality. However, reaccumulation occurred rapidly after far-red but slowly after red irradiation, indicating different modes of regulation of phytochrome expression after light-dark transitions depending on the light quality of the preceding irradiation. The wavelength dependency of the destruction rates was distinct from that of mustard, a close relative of Arabidopsis, and was explained on the basis of Pfr-induced Pr destruction and a simple kinetic two-step model. No dark reversion was detectable in the destruction kinetics after a red pulse. From these data we conclude that Arabidopsis phyA differs significantly in several aspects from other dicot phytochromes.     

Phytochrome A and Phytochrome B Have Overlapping but Distinct Functions in Arabidopsis Development

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. In Arabidopsis thaliana, there are genes encoding at least five phytochromes, and it is of interest to learn if the different phytochromes have overlapping or distinct functions. To address this question for two of the phytochromes in Arabidopsis, we have compared light responses of the wild type with those of a phyA null mutant, a phyB null mutant, and a phyA phyB double mutant. We have found that both phyA and phyB mutants have a deficiency in germination, the phyA mutant in far-red light and the phyB mutant in the dark. Furthermore, the germination defect caused by the phyA mutation in far- red light could be suppressed by a phyB mutation, suggesting that phytochrome B (PHYB) can have an inhibitory as well as a stimulatory effect on germination. In red light, the phyA phyB double mutant, but neither single mutant, had poorly developed cotyledons, as well as reduced red-light induction of CAB gene expression and potentiation of chlorophyll induction. The phyA mutant was deficient in sensing a flowering response inductive photoperiod, suggesting that PHYA participates in sensing daylength. In contrast, the phyB mutant flowered earlier than the wild type (and the phyA mutant) under all photoperiods tested, but responded to an inductive photoperiod. Thus, PHYA and PHYB appear to have complementary functions in controlling germination, seedling development, and flowering. We discuss the implications of these results for possible mechanisms of PHYA and PHYB signal transduction.