Low Fluence Response Research Articles

Investigating the Effects of Dark Period Light Exposure on Sex Expression In Female Cannabis sativa

Hermaphroditism in female plants is a common problem in the cannabis industry. Cannabis sativa florogenesis is dependent on photoperiod, and industry experts suspect disruptions to the photoperiod contribute to hermaphroditism. The goals of this report are to (i) determine the relationship between hermaphroditism and dark period light exposure in C. sativa, (ii) identify a causal mechanism that could explain female hermaphroditism, and (iii) recommend a course of action for indoor cultivators. A literature search suggested that hermaphroditism in C. sativacould be the result of a very low fluence response. To test this idea, 403 plants grown indoors were inspected for male flowers. The distance of each plant from the nearest grow room door—the proposed source of dark period light—was used to calculate relative dark period light intensity values for each plant. Linear regression analysis was used to assess the relationship between relative light intensity and males per plant. The average males per plant was 0.136, compared with an intercept coefficient of 0.125 (p=0.00197, R2=0.0162). Based on these results, proximity to a door does not affect hermaphroditism. Because the actual light intensities during dark periods in this study were likely too low to cause very low fluence responses, this study cannot confidently evaluate the relationship between dark period light exposure and hermaphroditism in C. sativa. Management at the study site would be advised not to spend resources addressing grow room door light leaks.

Two Distinct Molecular Types of Phytochrome A in Plants: Evidence of Existence and Implications for Functioning.

Phytochrome (phy) system in plants comprising a small number of phytochromes with phyA and phyB as major ones is responsible for acquiring light information in the red-far-red region of the solar spectrum. It provides optimal strategy for plant development under changing light conditions throughout all its life cycle beginning from seed germination and seedling establishment to fruiting and plant senescence. The phyA was shown to participate in the regulation of this cycle which is especially evident at its early stages. It mediates three modes of reactions-the very low and low fluence responses (VLFR and LFR) and the high irradiance responses (HIR). The phyA is the sole light receptor in the far-red spectral region responsible for plant's survival under a dense plant canopy where light is enriched with the far-red component. Its appearance is believed to be one of the main factors of plants' successful evolution. So far, it is widely accepted that one molecular phyA species is responsible for its complex functional manifestations. In this review, the evidence of the existence of two distinct phyA types-major, light-labile and soluble phyA' and minor, relatively light-stable and amphiphilic phyA″-is presented as what may account for the diverse modes of phyA action.

Phytochrome A in plants comprises two structurally and functionally distinct populations-water-soluble phyA' and amphiphilic phyA″.

Photoreceptor phytochrome A (phyA) plays a key role both in the individual development and in the evolution of higher plants. It acts in three distinct modes - far-red light-induced very low fluence responses (VLFRs), high irradiance responses (HIRs), and red/far-red-reversible low fluence responses (LFRs). Signal transduction from phyA includes its transportation from the cytoplasm into the nucleus and activation of light-responsive genes there. It is also active in the cytoplasm. Two types of phyA speckles were detected upon its light-induced nucleocytoplasmic partitioning and a fraction remained in the cytoplasm. In this review, we present a concept that this complex picture of the phyA action is due, at least partially, to the existence of two phyA types in the cell differing by the structure of the N-terminus, probably, by its serine phosphorylation. These are phosphorylated water-soluble phyA' and underphosphorylated ambiquitous phyA″ represented by two fractions - water-soluble and membrane-associated. From the analysis of the phyA pools' activity in the regulation of phyA synthesis, seed germination, seedling establishment, and (proto)chlorophyll biosynthesis it is concluded that phyA″ is responsible for the regulation of seed germination, whereas in seedlings phyA' mediates the VLFRs, and the water-soluble phyA″ fraction, the HIRs. The membrane-associated phyA″ is likely to be active in cytoplasmic photoregulatory events. Functional interaction between phyA and the defense-related hormone jasmonic acid is also considered.

Gas-Plasma-Activated Water Impact on Photo-Dependent Dormancy Mechanisms in Nicotiana tabacum Seeds

Seeds sense temperature, nutrient levels and light conditions to inform decision making on the timing of germination. Limited light availability for photoblastic species results in irregular germination timing and losses of population germination percentage. Seed industries are therefore looking for interventions to mitigate this risk. A growing area of research is water treated with gas plasma (GPAW), in which the formed solution is a complex consisting of reactive oxygen and nitrogen species. Gas plasma technology is widely used for sterilisation and is an emerging technology in the food processing industry. The use of the GPAW on seeds has previously led to an increase in germination performance, often attributed to bolstered antioxidant defence mechanisms. However, there is a limited understanding of how the solution may influence the mechanisms that govern seed dormancy and whether photoreceptor-driven germination mechanisms are affected. In our work, we studied how GPAW can influence the mechanisms that govern photo-dependent dormancy, isolating the effects at low fluence response (LFR) and very low fluence response (VLFR). The two defined light intensity thresholds affect germination through different phytochrome photoreceptors, PHYB and PHYA, respectively; we found that GPAW showed a significant increase in population germination percentage under VLFR and further described how each treatment affects key physiological regulators.

Analysis of Phytochrome-Dependent Seed Germination in Arabidopsis.

Light-dependent seed germination guarantees seedling proximity to the soil surface, enabling quick photosynthetic energy supply. While seedling hypocotyl length is mainly used in phytochrome physiological assays to determine the functional impact of photoreceptor point mutations, different intracellular localizations, or the function of signal transduction components, phytochrome-controlled seed germination offers a different, very sensitive tool to test the phytochrome photoreceptor network. Photon fluences as low as 1nmolm-2 are sufficient to elicit the phytochrome A (phyA)-dependent very low fluence response (VLFR), whereas higher fluences (> 10μmolm-2) are needed to elicit the phyB-controlled and phyB-photoreversible low fluence response (LFR). Taking advantage of the different sensitivities of both phytochromes to different light qualities and quantities, a screening protocol is presented to score germination under different light conditions.

Effects of Light and Autotoxicity on the Reproduction of Bidens pilosa L.: From Laboratory to the Field

In Taiwan, the good agricultural practices for Bidens pilosa L. (Cobbler’s pegs) had been established due to its pharmacology application. However, the reproduction of this species that may cause phytotoxicity to the subsequent crops has not been investigated extensively. We hypothesized that both the phytotoxicity and canopy shading conditions that were altered by agricultural practices might affect its seed reproduction. Three experiments from laboratory, pot and field were conducted under different light treatments and residue application rates to evaluate the light requirement and phytotoxicity on the germination of Cobbler’s pegs. The results showed that the germination in the laboratory, dark treatments was higher than that of light treatment while it was inhibited in the darkness in the pot experiments (24% of the light treatments). Moreover, some seeds in the pot experiments germinated in the far-red light (FR) pretreated dark treatments. This observation indicated that the germination response of the investigated plant might be a very low fluence response (VLFR). Results also showed that the autotoxicity enhanced the germination reduction in the FR pretreated dark treatment while increasing the residues buried in the field. Accordingly, both autotoxicity and canopy shading may inhibit the reproduction of Cobbler’s pegs, but the application method needs further study.

Characterization of phytochrome C functions in the control of de-etiolation and agronomic traits in rice

Although phytochrome A (phyA) and phyB have been functionally characterized, functions of phyC in rice growth and development have remained elusive because of the functional dependency of phyC on the phyB protein. In this study, we introduced PHYB(C364A), in which the chromophore attachment site cysteine 364 was converted to alanine, into the phyAphyB double mutant (aabb) and the phyAphyBphyC triple mutant (aabbcc) to produce PHYB(C364A)/aabb lines and PHYB(C364A)/aabbcc lines, respectively. PHYB(C364A)/aabbcc lines were insensitive to red light (R) and far-red light (FR), suggesting that PHYB(C364A) protein was biologically inactive. Functions of phyC were characterized using the PHYB(C364A)/aabb lines, without the functional interference of phyA or phyB. Phytochrome C responded to R and FR to trigger de-etiolation in the very-low-fluence response and low-fluence response in the PHYB(C364A)/aabb lines. Compared with the aabb mutant, seedlings of PHYB(C364A)/aabb lines showed higher chlorophyll content and reduced leaf angle. The PHYB(C364A)/aabb lines also showed a delayed heading date under long-day conditions. Phytochrome C-regulated agronomic traits were measured at the mature stage. The PHYB(C364A)/aabb lines showed significantly increased plant height, panicle length, grain number per main panicle, seed-setting rate, grain size, and grain weight, compared with those of the aabb mutant. Taken together, the present findings confirm that phyC perceives R and FR, and plays an important role in photomorphogenesis and yield determination in rice.

A New Perspective on the Effect of UV-B on l-Ascorbic Acid Metabolism in Cucumber Seedlings.

This study aimed to examine the effects of UV-B on AsA and gene expression in cucumber seedlings. Particular emphasis was placed on identifying genes that were responsive to UV-B to increase AsA levels and elucidate the key UV-B response pathway. We found that the activities of myo-inositol oxygenase (MIOX), galactono-1,4-lactone dehydrogenase (GLDH), ascorbate oxidase (AO), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR), and the transcript levels of CsMIOX1, CsGLDH, CsAO2, CsAO4, CsGR1, CsAPX5, and CsDHAR1 significantly increased with UV-B exposure. These observations indicate that UV-B induces the expression of genes involved in d-mannose/l-galactose and myo-inositol pathways and the ascorbate-glutathione system. Moreover, several genes related to the low and high UV-B fluence responses were considered. CsHY5 and CsMYB60 were involved with the low-fluence response and appeared to be responsive from 2 to 28 h. Together, these data show that these genes respond to UV-B to increase AsA levels through the low-fluence UV-B response pathway.

Reproductive Induction is a Far-Red High Irradiance Response that is Mediated by Phytochrome and PHYTOCHROME INTERACTING FACTOR in Marchantia polymorpha.

Land plants have evolved a series of photoreceptors to precisely perceive environmental information. Among these, phytochromes are the sole photoreceptors for red light (R) and far-red light (FR), and play pivotal roles in modulating various developmental processes. Most extant land plants possess multiple phytochromes that probably evolved from a single phytochrome in the common ancestor of land plants. However, the ancestral phytochrome signaling mechanism remains unknown due to a paucity of knowledge regarding phytochrome functions in basal land plants. It has recently been reported that Mpphy, a single phytochrome in the liverwort Marchantia polymorpha, regulates typical photoreversible responses collectively classified as low fluence response (LFR). Here, we show that Mpphy also regulates the gametangiophore formation analogous to the mode of action of the far-red high irradiance response (FR-HIR) in angiosperms. Our phenotypic analyses using mutant plants obtained by CRISPR/Cas9-based genome editing revealed that MpFHY1, an ortholog of FAR-RED ELONGATED HYPOCOTYL1, as well as Mpphy is critical for the FR-HIR signaling in M. polymorpha. In addition, knockout of MpPIF, a single PHYTOCHROME INTERACTING FACTOR gene in M. polymorpha, completely abolished the FR-HIR-dependent gametangiophore formation, while overexpression of MpPIF accelerated the response. FR-HIR-dependent transcriptional regulation was also disrupted in the Mppif mutant. Our findings suggest that plants had already acquired the FR-HIR signaling mediated by phytochrome and PIF at a very early stage during the course of land plant evolution, and that a single phytochrome in the common ancestor of land plants could mediate both LFR and FR-HIR.

Two molecular species of phytochrome A with distinct modes of action.

Adaptation of plants to environmental light conditions is achieved via operation of a highly complex photoreceptor apparatus. It includes the phytochrome system comprising phytochromes A and B (phyA and phyB) as the major components. phyA differs from phyB by several properties, including its ability to mediate all three photoresponse modes - the very low and low fluence responses (VLFR and LFR respectively) and the high irradiance responses (HIR), whereas phyB is responsible for LFR. This review discusses the uniqueness of phyA in terms of its structural and functional heterogeneity. The photoreceptor is presented in monocots and dicots by two native molecular species, phyA' and phyA'', differing by spectroscopic, photochemical and phenomenological properties. phyA differentiation into substates includes post-translational phosphorylation of a serine residue(s) at the N-terminal extension of the molecule with phyA' being the phosphorylated species and phyA'', dephosphorylated. They differ also by their mode of action, which depends on the cellular context. The current working hypothesis is that phyA' mediates VLFR and phyA'', HIR and LFR. The content and functional activity of the two pools are regulated by light and by phosphatase/kinase equilibrium and pH in darkness, what contributes to the fine-tuning of the phytochrome system. Detection of the native pools of the cryptogamic plant fern Adiantum capillus-veneris phy1 (phy1' and phy1'') similar to those of phyA suggests that the structural and functional heterogeneity of phyA is not a unique phenomenon and may have arisen earlier in the molecular evolution of the phytochrome system than the appearance of the angiosperm phytochromes.

Biological activity and dimerization state of modified phytochrome A proteins.

To assess potential physical interactions of type I phyA with the type II phyB-phyE phytochromes in vivo, transgenes expressing fusion gene forms of phyA were introduced into the Arabidopsis phyA mutant background. When a single c-Myc (myc) epitope is added to either the N- or C-terminus of phyA, the constructs completely complement phyA mutant phenotypes. However, addition of larger tags, such as six consecutive myc epitopes or the yellow fluorescent protein sequence, result in fusion proteins that show reduced activity. All the tagged phyA proteins migrate as dimers on native gels and co-immunoprecipitation reveals no binding interaction of phyA to any of the type II phys in the dark or under continuous far-red light. Dimers of the phyA 1–615 amino acid N-terminal photosensory domain (NphyA), generated in vivo with a yeast GAL4 dimerization domain and attached to a constitutive nuclear localization sequence, are expressed at a low level and, although they cause a cop phenotype in darkness and mediate a very low fluence response to pulses of FR, have no activity under continuous FR. It is concluded that type I phyA in its Pr form is present in plants predominantly or exclusively as a homodimer and does not stably interact with type II phys in a dimer-to-dimer manner. In addition, its activity in mediating response to continuous FR is sensitive to modification of its N- or C-terminus.

Low-level lasers on microRNA and uncoupling protein 2 mRNA levels in human breast cancer cells

MicroRNA is short non-coding RNA and is a mediator of post-transcriptional regulation of gene expression. In addition, uncoupling proteins (UCPs) regulate thermogenesis, metabolic and energy balance, and decrease reactive oxygen species production. Both microRNA and UCP2 expression can be altered in cancer cells. At low power, laser wavelength, frequency, fluence and emission mode deternube photobiological responses, which are the basis of low-level laser therapy. There are few studies on miRNA and UCP mRNA levels after low-level laser exposure on cancer cells. In this work, we evaluate the micrRNA (mir-106b and mir-15a) and UCP2 mRNA levels in human breast cancer cells exposed to low-level lasers. MDA-MB-231 human breast cancer cells were exposed to low-level red and infrared lasers, total RNA was extracted for cDNA synthesis and mRNA levels by real time quantitative polymerase chain reaction were evaluated. Data show that mir-106b and mir-15a relative levels are not altered, but UCP2 mRNA relative levels are increased in MDA-MB-231 human breast cancer cells exposed to low-level red and infrared lasers at fluences used in therapeutic protocols.

Phytochrome Signaling Is Mediated by PHYTOCHROME INTERACTING FACTOR in the Liverwort Marchantia polymorpha.

Phytochromes are red light (R) and far-red light (FR) receptors that play important roles in many aspects of plant growth and development. Phytochromes mainly function in the nucleus and regulate sets of genes by inhibiting negatively acting basic helix-loop-helix transcription factors named PHYTOCHROME INTERACTING FACTORs (PIFs) in Arabidopsis thaliana Although R/FR photoreversible responses and phytochrome genes are well documented in diverse lineages of plants, the extent to which phytochrome signaling is mediated by gene regulation beyond angiosperms remains largely unclear. Here, we show that the liverwort Marchantia polymorpha, an emerging model basal land plant, has only one phytochrome gene, Mp-PHY, and only one PIF gene, Mp-PIF These genes mediate typical low fluence responses, which are reversibly elicited by R and FR, and regulate gene expression. Mp-phy is light-stable and translocates into the nucleus upon irradiation with either R or FR, demonstrating that the single phytochrome Mp-phy exhibits combined biochemical and cell-biological characteristics of type I and type II phytochromes. Mp-phy photoreversibly regulates gemma germination and downstream gene expression by interacting with Mp-PIF and targeting it for degradation in an R-dependent manner. Our findings suggest that the molecular mechanisms for light-dependent transcriptional regulation mediated by PIF transcription factors were established early in land plant evolution.

Synergistic Effect of Ionization and Displacement Defects in NPN Transistors Induced by 40-MeV Si Ion Irradiation With Low Fluence

Based on 3DG110 transistors and special gated NPN (GNPN) transistors, the characteristic of the synergistic effect between ionization and displacement defects induced by 40-MeV silicon (Si) ions with low fluence was investigated in terms of deep-level transient spectroscopy (DLTS). Nonlinear relationship, produced by the synergistic effect, between low fluence and radiation response can be obviously observed in the bipolar junction transistors (BJTs) under the exposure of 40-MeV Si ions. The DLTS signals of displacement defect centers and oxide-trapped charges are detected in the base–collector junction of the 3DG110 transistors. Meanwhile, based on the GNPN transistors, the DLTS signals of interface traps and displacement defects are measured in the base–collector junction. By comparison of the change in electrical parameters and the DLTS signals, it is found that the interface traps induce an enhanced effect to displacement defects in the base–collector junction of NPN BJT, whereas the oxide-trapped charge can suppress the DLTS signals of deep-level centers to a certain extent. Compared with the suppression action, the enhanced effect rising from ionization damage gives more contribution to the displacement damage. Moreover, the bias used during DLTS measurements can influence the characteristics of DLTS signals caused by oxide-trapped charge and interface traps. With increasing bias, both the height and temperature of ionization defect peaks in the DLTS spectra will increase, illustrating that concentration and energy level of these defects are raised.

Atmospheric moisture controls far-red irradiation: a probable impact on the phytochrome

Abstract It is commonly accepted that an important role of the phytochrome lies in signalling the proximity of competing plants. However, not all photoresponses conveyed by the phytochrome can be explained by the competition only. Because a better description of the natural variability of solar spectral irradiance is necessary to recognize the other roles of the phytochrome, long-lasting spectroradiometric measurements have been performed. Special attention has been paid to the relations between the far-red and red bands of the solar spectrum, which have an impact on the phytochrome. The effect of atmospheric moisture on far-red irradiance (attenuated in the 720 nm band of water vapour absorption) is described. The far-red irradiance, active in the ‘high irradiance response’ of the phytochrome, and the red/far-red ratio, important for the ‘low fluence response’, may vary very strongly relative to the atmospheric moisture. Together with other facts known from photophysiology, the results of the measurements enabled us to formulate a thesis that the phytochrome monitors the amount of water vapour and opens appropriate metabolic pathways to cope with the danger of drought. The recognition of this novel role of the phytochrome might broaden the knowledge in the area of plant photomorphogenesis and ecology.

Phototropin 1 and dim-blue light modulate the red light de-etiolation response

Light signals regulate seedling morphological changes during de-etiolation through the coordinated actions of multiple light-sensing pathways. Previously we have shown that red-light-induced hypocotyl growth inhibition can be reversed by addition of dim blue light through the action of phototropin 1 (phot1). Here we further examine the fluence-rate relationships of this blue light effect in short-term (hours) and long-term (days) hypocotyl growth assays. The red stem-growth inhibition and blue promotion is a low-fluence rate response, and blue light delays or attenuates both the red light and far-red light responses. These de-etiolation responses include blue light reversal of red or far-red induced apical hook opening. This response also requires phot1. Cryptochromes (cry1 and cry2) are activated by higher blue light fluence-rates and override phot1's influence on hypocotyl growth promotion. Exogenous application of auxin transport inhibitor naphthylphthalamic acid abolished the blue light stem growth promotion in both hypocotyl growth and hook opening. Results from the genetic tests of this blue light effect in auxin transporter mutants, as well as phytochrome kinase substrate mutants indicated that aux1 may play a role in blue light reversal of red light response. Together, the phot1-mediated adjustment of phytochrome-regulated photomorphogenic events is most robust in dim blue light conditions and is likely modulated by auxin transport through its transporters.

From UVR8 to flavonol synthase: UV-B-induced gene expression in Sauvignon blanc grape berry.

The aim of this research was to determine the effect of development and UV-B on flavonols and the regulation of gene activity in Vitis vinifera L. var. Sauvignon blanc grapes. Particular emphasis was placed on gene activity associated with the low and high fluence UV-B responses. Flavonols, particularly quercetin and kaempferol glycosides, increased substantially upon fruit exposure due to UV-B, with spatial analysis locating the changes to the berry skin. Of five VvFLS genes in grapes, two (VvFLS4 and 5) were found to be transcriptionally active, with VvFLS4 also being responsive to UV-B but VvFLS5 was not. Of the transcription factors known to regulate FLS (VvMYB12, VvMYCA1 and VvWDRs), only VvMYB12 was found to be responsive to UV-B. A number of candidate genes associated with the low and high UV-B fluence responses were also studied (VvUVR8, VvHY5, VvCOP1 and VvCHS; PR genes and VvMAPK3; respectively). The genes associated with the low fluence response exhibited transcriptional regulation in line with reports from other species, while the PR genes and VvMAPK3 only appeared to be responsive in a high UV-B fluence environment. Together, these data supports the view flavonol biosynthesis in grape is stimulated predominantly through the low fluence UV-B response pathway.

Striga hermonthica MAX2 restores branching but not the Very Low Fluence Response in the Arabidopsis thaliana max2 mutant

Seed germination of Striga spp. (witchweeds), one of the world's most destructive parasitic weeds, cannot be induced by light but is specifically induced by strigolactones. It is not known whether Striga uses the same components for strigolactone signaling as host plants, whether it has endogenous strigolactone biosynthesis and whether there is post-germination strigolactone signaling in Striga. Strigolactones could not be detected in in vitro grown Striga, while for host-grown Striga, the strigolactone profile is dominated by a subset of the strigolactones present in the host. Branching of in vitro grown Striga is affected by strigolactone biosynthesis inhibitors. ShMAX2, the Striga ortholog of Arabidopsis MORE AXILLARY BRANCHING 2 (AtMAX2) - which mediates strigolactone signaling - complements several of the Arabidopsis max2-1 phenotypes, including the root and shoot phenotype, the High Irradiance Response and the response to strigolactones. Seed germination of max2-1 complemented with ShMAX2 showed no complementation of the Very Low Fluence Response phenotype of max2-1. Results provide indirect evidence for ShMAX2 functions in Striga. A putative role of ShMAX2 in strigolactone-dependent seed germination of Striga is discussed.

Dynamics of the processes leading to the acquisition of sensitivity to very low fluence of photons inDatura feroxseeds

AbstractSoil tillage operations stimulate germination of buried seeds in cultivated lands, allowing them to perceive light as a germination-promoting factor. The time of burial and the effect of changing environmental factors affect the physiological state of the seeds, which may lead to an extreme light-sensitivity and very low fluence response (VLFR) through phytochrome A. This paper describes the influence of the progressive process of dormancy breakage, which is accompanied by the acquisition of extreme light-sensitivity, on processes associated with endosperm weakening and embryo growth potential in the VLFR-mediated promotion ofDatura feroxseed germination. Our results show that endosperm weakening is mainly limited by β-mannosidase enzyme activity after far-red light stimulation, which is highly dependent on the dormancy level of the seeds. In addition, stimulation of the embryo growth potential by far-red irradiation did not require an extreme light-sensitivity to very low fluence of photons to reach its maximum response, and it was not completely correlated with expansin gene expression in the embryo. Our work indicates that responses of endosperm weakening and embryo growth potential to far-red irradiation, dependent on dormancy level, have different requirements for stimulation by the signalling network initiated by phytochrome A during the course of the very low fluence response inDatura feroxseeds.

MAP-kinase activity in etiolated Cucumis sativus cotyledons: The effect of red and far-red light irradiation

Phytochrome (phy) signalling in plants may be transduced through protein phosphorylation. Mitogen-activated protein kinase (MAP-kinase, MAPK) activity and the effect of R (red) and FR (far-red) light irradiation on MAPK activity were studied in etiolated Cucumis sativus L. cotyledons. By in vitro protein phosphorylation and in-gel assays with myelin basic protein (MBP), a protein band (between 48 and 45 kDa) with MAPK-like activity was detected. The addition to the phosphorylation buffer of specific protein phosphatase (PTP) inhibitors (Na(3)VO(4) and NaF) and genistein, apigenin or PD98059 as MAPK inhibitors allowed us to confirm the MAPK activity of the protein band. Irradiation of etiolated cotyledons with FR light for 5, 10 or 60 min rapidly and transiently stimulated the MAPK activity of the protein band. This suggests that there was a very low fluence response (VLFR) of phys. In addition, 15 min of R light irradiation or a sequential treatment of 15 min of R plus 5 min of FR also increased MAPK activity. The stimulatory effect of R light was also attributed to the same photoreceptor, which suggests that MAPKs are involved in phytochrome signal transduction. Protein immunoprecipitation and immunoblotting analysis with the polyclonal antibody anti-pERK1/2 (Tyr 204) and the monoclonal antibody anti-phosphotyrosine PY20 allowed us to recognize the above mentioned protein band as two proteins with molecular masses (M(r)) of approximately 47 and 45 kDa, and MAPK activity. The biochemical and immunological properties showed by the proteins detected indicated that they were members of the MAPK family phosphorylated in tyrosine residues.