Very Low Fluence Response Research Articles

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.

Light perception and signalling by phytochrome A

In etiolated seedlings, phytochrome A (phyA) mediates very-low-fluence responses (VLFRs), which initiate de-etiolation at the interphase between the soil and above-ground environments, and high-irradiance responses (HIR), which complete de-etiolation under dense canopies and require more sustained activation with far-red light. Light-activated phyA is transported to the nucleus by FAR-RED ELONGATED HYPOCOTYL1 (FHY1). The nuclear pool of active phyA increases under prolonged far-red light of relatively high fluence rates. This condition maximizes the rate of FHY1-phyA complex assembly and disassembly, allowing FHY1 to return to the cytoplasm to translocate further phyA to the nucleus, to replace phyA degraded in the proteasome. The core signalling pathways downstream of nuclear phyA involve the negative regulation of CONSTITUTIVE PHOTOMORPHOGENIC 1, which targets for degradation transcription factors required for photomorphogenesis, and PHYTOCHROME-INTERACTING FACTORs, which are transcription factors that repress photomorphogenesis. Under sustained far-red light activation, released FHY1 can also be recruited with active phyA to target gene promoters as a transcriptional activator, and nuclear phyA signalling activates a positive regulatory loop involving BELL-LIKE HOMEODOMAIN 1 that reinforces the HIR.

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.

OWL1 Is a Phytochrome A Signaling Component Dedicated to the Very Low Fluence Response

In Arabidopsis thaliana , there are five phytochromes that sense red (R) and far-red (FR) light (reviewed in [Bae and Choi, 2008][1]). Phytochrome A (phyA) is the major phytochrome in etiolated seedlings and plays roles in the very low fluence response (VLFR), high irradiance responses (HIRs), and,

Bell-like homeodomain selectively regulates the high-irradiance response of phytochrome A

Plant responses mediated by phytochrome A display a first phase saturated by transient light signals and a second phase requiring sustained excitation with far-red light (FR). These discrete outcomes, respectively so-called very-low-fluence response (VLFR) and high-irradiance response (HIR), are appropriate in different environmental and developmental contexts but the mechanisms that regulate the switch remain unexplored. Promoter analysis of a light-responsive target gene revealed a motif necessary for HIR but not for VLFR. This motif is required for binding of the Bell-like homeodomain 1 (BLH1) to the promoter in in vitro and in yeast 1-hybrid experiments. Promoter substitutions that increased BLH1 binding also enhanced HIR. blh1 mutants showed reduced responses to continuous FR and to deep canopy shadelight, but they retained normal responses to pulsed FR or red light and unfiltered sunlight. BLH1 enhanced BLH1 expression and its promotion by FR. We conclude that BLH1 specifically regulates HIR and not VLFR of phytochrome A.

A Rice Phytochrome A in Arabidopsis: The Role of the N-terminus under red and far-red light

The phytochrome (phy)A and phyB photoreceptors mediate three photobiological response modes in plants; whereas phyA can mediate the very-low-fluence response (VLFR), the high-irradiance response (HIR) and, to some extent, the low fluence response (LFR), phyB and other type II phytochromes only mediate the LFR. To investigate to what level a rice phyA can complement for Arabidopsis phyA or phyB function and to evaluate the role of the serine residues in the first 20 amino acids of the N-terminus of phyA, we examined VLFR, LFR, and HIR responses in phyB and phyAphyB mutant plants transformed with rice PHYA cDNA or a mutant rice PHYA cDNA in which the first 10 serine residues were mutated to alanines (phyA SA). Utilizing mutants without endogenous phyB allowed the evaluation of red-light-derived responses sensed by the rice phyA. In summary, the WT rice phyA could complement VLFR and LFR responses such as inhibition of hypocotyl elongation under pulses of FR or continuous R light, induction of flowering and leaf expansion, whereas the phyA SA was more specific for HIR responses (e.g. inhibition of hypocotyl elongation and anthocyanin accumulation under continuous far-red light). As the N-terminal serines can no longer be phosphorylated in the phyA SA mutant, this suggests a role for phosphorylation discriminating between the different phyA-dependent responses. The efficacy of the rice phyA expressed in Arabidopsis was dependent upon the developmental age of the plants analyzed and on the physiological response, suggesting a stage-dependent downstream modulation of phytochrome signaling.

The very-low-fluence and high-irradiance responses of the phytochromes have antagonistic effects on germination, mannan-degrading activities, and DfGA3ox transcript levels in Datura ferox seeds

Seed germination can be promoted by the modes of action of two of the phytochromes: the low-fluence response (LFR), which is the classical red (R)-far-red (FR) reversible response and the very-low-fluence response (VLFR) that can be saturated by extremely low levels of Pfr, which can be elicited by a saturating FR pulse. The Datura ferox seed population used in this work had acquired the capacity to germinate through a VLFR after pretreatment in a water-saturated atmosphere (WSA) at constant 25 degrees C. After 12 d in WSA germination after a FR pulse was 82%, while it was less than 10% in darkness. It was found that the VLFR of germination is associated with increments in the embryo growth potential (EGP) and in the activity of two enzymes related to the weakening of the micropylar region of the endosperm (ME); endo-beta-mannanase and beta-mannosidase. The FR pulse also significantly stimulated the expression of DfGA3ox, a GA 3beta-hydroxylase, suggesting that the promotion of germination by the VLFR is associated with an increase in the synthesis of active gibberellins. The promotive action of the VLFR on germination is reduced when the FR pulse is immediately followed by a continuous FR treatment for 24 h (FRc). The effect of FRc cannot be reproduced by hourly FR pulses during the same period, showing that the antagonistic effect of FRc is a high-irradiance response (HIR). The action of the HIR in germination is associated with a decrease of both the mannan-degrading enzyme activity and the expression of DfMan in the ME, whereas no changes in the EGP were observed. The HIR also inhibits the accumulation of DfGA3ox in embryos, indicating that its action on germination is mediated, at least in part, through the modulation of active GA contents in seeds. This is the first report of a gene that participates in the VLFR-HIR antagonism in seeds.

Blue Rhythms Between GIGANTEA and Phytochromes

GIGANTEA (GI) is involved in the promotion of flowering by long days, in light input to the circadian clock, and in seedling de-etiolation under continuous red light or blue light but not under continuous far-red light (FR). Since red-light effects are mediated largely by phytochrome B and those of FR by phytochrome A (phyA), GI was considered not to affect phyA signaling. However, recent observations using brief FR pulses indicate that GI does affect the very-low-fluence response (VLFR) and not the high-irradiance response (HIR) pathway of phyA. Seed germination, seedling de-etiolation and gene expression showed rhythmic sensitivity to FR pulses. GI affected the magnitude but not the rhythmic pattern of the responses, indicating that GI regulation of phyA signaling does not derive from its effects on the clock. Here we show that despite de above divergence between VLFR and HIR, both pathways require the bZip transcription factor HY5. Furthermore, the blue-light phenotype of gi is not mediated by phyA, indicating a role of GI in the control of cryptochrome signaling. These findings illustrate features of plant signaling networks.

Light environment in pre- and post-dehiscent fruits affects seed germination in Calotropis procera

Fruits in Calotropis procera can be distinguished into five discrete but contiguous stages on the basis of diameter and seed color. Seeds from dehisced fruits at stage V germinated >80% on moist substratum in darkness. This was rather unexpected because the seeds developed and matured in an FR-enriched microenvironment (R:FR ratio ∼0.3) of the chlorophyll-containing maternal tissue and displayed low-fluence response (LFR) mode of phytochrome action. In contrast to >80% dark-germinating seeds from dehisced fruits at stage V, about 50% seeds from undehisced fruits at that stage were dark germinating, whereas another 30% seeds required light for germination. The light-requiring fraction of the seed population did not only respond to a very low-fluence R and to a short FR pulse, but also lacked R–FR reversibility thereby indicating to a very low-fluence response (VLFR) mode of phytochrome action. The present study reporting VLFR to non-dormant seed state transition in C. procera suggested that the state of phytochrome and the subsequent seed germination response in dry-seeded species, besides being determined by the light environment immediately before maturation drying, might also be regulated by a post-dehiscence light signal.

Phytochrome B and shade signals regulate phytochrome A expression

The control of phytochrome A expression at the protein and mRNA levels was investigated in wild‐type and phyB‐1 mutant sorghum (Sorghum bicolor[L.] Moench). PHYA mRNA abundance follows a diurnal rhythm in both genotypes, with maximal accumulation near the latter part of the light period. PHYA mRNA is more abundant in the phyB‐1 mutant. The level of PHYA message correlates with both R : FR and photon flux density in wild‐type, but only with photon flux density in the phyB‐1 mutant. The differences in mRNA abundance are reflected in the level of phyA protein, which is elevated in the phyB‐1 mutant and accumulates under low photon flux density. During de‐etiolation, PHYA message accumulation is initially repressed solely by a very low fluence response (VLFR) presumably mediated by phyA. The phyB‐mediated low fluence response maintains the repression of accumulation past the time controlled by the VLFR. With repetitive photoperiods, the transition from the etiolated growth form to autotrophic competency is accompanied by a transition from light‐induced reduction of PHYA mRNA abundance to enhanced accumulation during the light period. The loss of phyB function allows partial de‐repression of PHYA message accumulation under repetitive photoperiods, resulting in plants deficient in phyB but enriched in phyA. The modification of PHYA mRNA and protein levels in the phyB‐1 mutant documented in this study may help clarify the molecular basis of the phyB‐1 phenotype. The tailoring of phyA abundance in wild‐type to the time of day and shade signals suggests a plastic role for this pigment in controlling development in light‐grown plants.

Changes in the light sensitivity of buried Polygonum aviculare seeds in relation to cold‐induced dormancy loss: development of a predictive model

The effect of cold (stratification) temperature on changes in the sensitivity of Polygonum aviculare seeds to light was investigated. Seeds buried in pots were stored under stratification temperatures (1.6, 7 and 12 degrees C) for 137 d. Seeds exhumed at regular intervals during storage were exposed to different light treatments. Germination responses obtained for seeds exposed to different light treatments and stratification temperatures were used to develop a model to predict the sensitivity of buried seeds to light. Seed sensitivity to light increased as dormancy loss progressed, showing the successive acquisition of low-fluence responses (LFR), very low-fluence responses (VLFR), and the loss of the light requirement for germination for a fraction of the seed population. These changes were inversely correlated to stratification temperature, allowing the use of a thermal time index to relate observed changes in seed light sensitivity to stratification temperature. The rate of increase in sensitivity of P. aviculare seeds to light during stratification is inversely correlated to soil temperature, and these changes in light sensitivity could be predicted in relation to temperature using thermal-time models.

A growth regulatory loop that provides homeostasis to phytochrome a signaling.

Phytochrome kinase substrate1 (PKS1) is a cytoplasmic protein that interacts physically with, and is phosphorylated by, the plant photoreceptor phytochrome. Here, we show that light transiently increases PKS1 mRNA levels and concentrates its expression to the elongation zone of the hypocotyl and root. This response is mediated by phytochrome A (phyA) acting in the very low fluence response (VLFR) mode. In the hypocotyl, PKS1 RNA and protein accumulation are maintained only under prolonged incubation in far-red light, the wavelength that most effectively activates phyA. Null mutants of PKS1 and its closest homolog, PKS2, show enhanced phyA-mediated VLFR. Notably, a pks1 pks2 double mutant has no phenotype, whereas overexpression of either PKS1 or PKS2 results in the same phenotype as the pks1 or pks2 single null mutant. We propose that PKS1 and PKS2 are involved in a growth regulatory loop that provides homeostasis to phyA signaling in the VLFR. In accordance with this idea, PKS1 effects are larger in the pks2 background (and vice versa). Moreover, the two proteins can interact with each other, and PKS2 negatively regulates PKS1 protein levels specifically under VLFR conditions.

The serine-rich N-terminal domain of oat phytochrome a helps regulate light responses and subnuclear localization of the photoreceptor.

Phytochrome (phy) A mediates two distinct photobiological responses in plants: the very-low-fluence response (VLFR), which can be saturated by short pulses of very-low-fluence light, and the high-irradiance response (HIR), which requires prolonged irradiation with higher fluences of far-red light (FR). To investigate whether the VLFR and HIR involve different domains within the phyA molecule, transgenic tobacco (Nicotiana tabacum cv Xanthi) and Arabidopsis seedlings expressing full-length (FL) and various deletion mutants of oat (Avena sativa) phyA were examined for their light sensitivity. Although most mutants were either partially active or inactive, a strong differential effect was observed for the Delta6-12 phyA mutant missing the serine-rich domain between amino acids 6 and 12. Delta6-12 phyA was as active as FL phyA for the VLFR of hypocotyl growth and cotyledon unfolding in Arabidopsis, and was hyperactive in the VLFR of hypocotyl growth and cotyledon unfolding in tobacco, and the VLFR blocking subsequent greening under white light in Arabidopsis. In contrast, Delta6-12 phyA showed a dominant-negative suppression of HIR in both species. In hypocotyl cells of Arabidopsis irradiated with FR phyA:green fluorescent protein (GFP) and Delta6-12 phyA:GFP fusions localized to the nucleus and coalesced into foci. The proportion of nuclei with abundant foci was enhanced by continuous compared with hourly FR provided at equal total fluence in FL phyA:GFP, and by Delta6-12 phyA mutation under hourly FR. We propose that the N-terminal serine-rich domain of phyA is involved in channeling downstream signaling via the VLFR or HIR pathways in different cellular contexts.

Missense mutation in the PAS2 domain of phytochrome A impairs subnuclear localization and a subset of responses.

Phytochrome A signaling shows two photobiologically discrete outputs: so-called very-low-fluence responses (VLFR) and high-irradiance responses (HIR). By modifying previous screening protocols, we isolated two Arabidopsis mutants retaining VLFR and lacking HIR. Phytochrome A negatively or positively regulates phytochrome B signaling, depending on light conditions. These mutants retained the negative but lacked the positive regulation. Both mutants carry the novel phyA-302 allele, in which Glu-777 (a residue conserved in angiosperm phytochromes) changed to Lys in the PAS2 motif of the C-terminal domain. The phyA-302 mutants showed a 50% reduction in phytochrome A levels in darkness, but this difference was compensated for by greater stability under continuous far-red light. phyA-302:green fluorescent protein fusion proteins showed normal translocation from the cytosol to the nucleus under continuous far-red light but failed to produce nuclear spots, suggesting that nuclear speckles could be involved in HIR signaling and phytochrome A degradation. We propose that the PAS2 domain of phytochrome A is necessary to initiate signaling in HIR but not in VLFR, likely via interaction with a specific partner.

SPA1, a component of phytochrome A signal transduction, regulates the light signaling current.

Mutations in a component of phytochrome A (phyA)-specific light signal transduction, SPA1, result in enhanced responsiveness of Arabidopsis seedlings to red and far-red light. Here, we have examined the effects of spa1 mutations on the two known modes of phyA function, the high-irradiance responses (HIRs) to continuous irradiation with far-red light and the very-low-fluence responses (VLFRs) to inductive pulses of light that establish only a small proportion of active phyA. spa1 mutants exhibited an enhanced VLFR under hourly pulses of far-red light for hypocotyl growth inhibition, cotyledon unfolding, anthocyanin accumulation, block of greening in subsequent white light and negative regulation of phyB signaling. We provide evidence that the phenotype of spa1 mutants in red light is also caused by an increase in the VLFR. Taken together, our results indicate that light-induced hypocotyl growth inhibition in spa1 mutants is primarily due to a VLFR. While wild-type seedlings required hourly pulses of far-red light to induce a VLFR, infrequent irradiation with far-red pulses (every 12 h) was sufficient to induce a strong VLFR of hypocotyl elongation in spa1 mutants. This shows that the effect of the VLFR was more persistent in spa1 mutants than in the wild type. We, therefore, propose that SPA1 has an important function in reducing the persistence of phyA signaling. spa1 mutations also enhanced the HIRs of anthocyanin accumulation and of phyA-mediated responsivity amplification towards phyB. Thus, our results suggest that spa1 mutations amplify both the phyA-mediated VLFR and the HIR.

Brassinosteroid Mutants Uncover Fine Tuning of Phytochrome Signaling

Phytochromes (phy) A and B provide higher plants the ability to perceive divergent light signals. phyB mediates red/far-red light reversible, low fluence responses (LFR). phyA mediates both very-low-fluence responses (VLFR), which saturate with single or infrequent light pulses of very low fluence, and high irradiance responses (HIR), which require sustained activation with far-red light. We investigated whether VLFR, LFR, and HIR are genetically coregulated. The Arabidopsis enhanced very-low-fluence response1 mutant, obtained in a novel screening under hourly far-red light pulses, showed enhanced VLFR of hypocotyl growth inhibition, cotyledon unfolding, blocking of greening, and anthocyanin synthesis. However, eve1 showed reduced LFR and HIR. eve1 was found allelic to the brassinosteroid biosynthesis mutant dim/dwarf1. The analysis of both the brassinosteroid mutant det2 in the Columbia background (where VLFR are repressed) and the phyA eve1 double mutant indicates that the negative effect of brassinosteroid mutations on LFR requires phyA signaling in the VLFR mode but not the expression of the VLFR. Under sunlight, hypocotyl growth of eve1 showed little difference with the wild type but failed to respond to canopy shadelight. We propose that the opposite regulation of VLFR versus LFR and HIR could be part of a context-dependent mechanism of adjustment of sensitivity to light signals.