Cryptochromes and phytochromes synergistically regulate Arabidopsis root greening under blue light

To increase their fitness, plants sense ambient light conditions and modulate their developmental processes by utilizing multiple photoreceptors such as phytochrome, cryptochrome and phototropin. Even roots, which are normally not exposed to light, express photoreceptors and can respond to light by developing chloroplasts. In the present study, root greening was observed in Arabidopsis thaliana. Seedlings were grown under monochromatic light and chlorophyll levels in the roots were determined. It was found that blue light was far more effective at inducing chloroplast development in Arabidopsis roots than was red light, and this response was under the control of a strong synergistic interaction between phytochromes and cryptochromes. As expected, the cry1 mutant was deficient in this response. Interestingly, the phyAphyB double mutant failed to respond to blue light under these conditions. This strongly suggests that either phytochrome A or phytochrome B, in addition to cryptochrome, was required for this blue light response. It was further demonstrated that the expression of photosynthetic genes was regulated in the same way. Dichromatic irradiation experiments indicated that this interaction depends on the level of phyB P(FR). Analysis of the cop1, det1 and hy5 mutants indicated that the corresponding factors were involved in the response.     

The Arabidopsis flavin-dependent monooxygenase FMO1 is an essential component of biologically induced systemic acquired resistance

Upon localized attack by necrotizing pathogens, plants gradually develop increased resistance against subsequent infections at the whole-plant level, a phenomenon known as systemic acquired resistance (SAR). To identify genes involved in the establishment of SAR, we pursued a strategy that combined gene expression information from microarray data with pathological characterization of selected Arabidopsis (Arabidopsis thaliana) T-DNA insertion lines. A gene that is up-regulated in Arabidopsis leaves inoculated with avirulent or virulent strains of the bacterial pathogen Pseudomonas syringae pv maculicola (Psm) showed homology to flavin-dependent monooxygenases (FMO) and was designated as FMO1. An Arabidopsis knockout line of FMO1 proved to be fully impaired in the establishment of SAR triggered by avirulent (Psm avrRpm1) or virulent (Psm) bacteria. Loss of SAR in the fmo1 mutants was accompanied by the inability to initiate systemic accumulation of salicylic acid (SA) and systemic expression of diverse defense-related genes. In contrast, responses at the site of pathogen attack, including increases in the levels of the defense signals SA and jasmonic acid, camalexin accumulation, and expression of various defense genes, were induced in a similar manner in both fmo1 mutant and wild-type plants. Consistently, the fmo1 mutation did not significantly affect local disease resistance toward virulent or avirulent bacteria in naive plants. Induction of FMO1 expression at the site of pathogen inoculation is independent of SA signaling, but attenuated in the Arabidopsis eds1 and pad4 defense mutants. Importantly, FMO1 expression is also systemically induced upon localized P. syringae infection. This systemic up-regulation is missing in the SAR-defective SA pathway mutants sid2 and npr1, as well as in the defense mutant ndr1, indicating a close correlation between systemic FMO1 expression and SAR establishment. Our findings suggest that the presence of the FMO1 gene product in systemic tissue is critical for the development of SAR, possibly by synthesis of a metabolite required for the transduction or amplification of a signal during the early phases of SAR establishment in systemic leaves.     

Phytochrome regulation of pea phototropin

Type 1 phototropin, one of the blue light receptors responsible for phototropism, is encoded in peas by at least two genes, PsPHOT1A and PsPHOT1B (formerly PsPK4 and PsPK5), both of which are more similar to Arabidopsis PHOT1 than to Arabidopsis PHOT2. We show here that PsPHOT1B encodes a full-length phototropin, whose expression pattern suggests that Psphot1b is the predominant phot1-type phototropin in etiolated seedlings. The gene encoding the other type 1 phototropin, PsPHOT1A, is expressed at low levels, with its highest levels in the leaves and stems of more mature, light-grown plants. Studies with phyA, phyB and the phyAphyB double mutants show that phyA and phyB have partially redundant roles in the reduction of PsPHOT1B expression under red light.     

Cryptochrome‐mediated hypocotyl phototropism was regulated antagonistically by gibberellic acid and sucrose in Arabidopsis

Both phototropins(phot1 and phot2) and cryptochromes(cry1 and cry2) were proven as the Arabidopsis thaliana blue light receptors. Phototropins predominately function in photomovement, and cryptochromes play a role in photomorphogenesis. Although cryptochromes have been proposed to serve as positive modulators of phototropic responses, the underlying mechanism remains unknown. Here, we report that depleting sucrose from the medium or adding gibberellic acids(GAs) can partially restore the defects in phototropic curvature of the phot1 phot2 double mutants under high-intensity blue light; this restoration does not occur in phot1 phot2 cry1 cry2 quadruple mutants and nph3(nonphototropic hypocotyl 3) mutants which were impaired phototropic response in sucrose-containing medium. These results indicate that GAs and sucrose antagonistically regulate hypocotyl phototropism in a cryptochromes dependent manner, but it showed a crosstalk with phototropin signaling on NPH3.Furthermore, cryptochromes activation by blue light inhibit GAs synthesis, thus stabilizing DELLAs to block hypocotyl growth, which result in the higher GAs content in the shade side than the lit side of hypocotyl to support the asymmetric growth of hypocotyl. Through modulation of the abundance of DELLAs by sucrose depletion or added GAs, it revealed that cryptochromes have a function in mediating phototropic curvature.     

Action spectrum for cryptochrome-dependent hypocotyl growth inhibition in Arabidopsis

Cryptochrome blue-light photoreceptors are found in both plants and animals and have been implicated in numerous developmental and circadian signaling pathways. Nevertheless, no action spectrum for a physiological response shown to be entirely under the control of cryptochrome has been reported. In this work, an action spectrum was determined in vivo for a cryptochrome-mediated high-irradiance response, the blue-light-dependent inhibition of hypocotyl elongation in Arabidopsis. Comparison of growth of wild-type, cry1cry2 cryptochrome-deficient double mutants, and cryptochrome-overexpressing seedlings demonstrated that responsivity to monochromatic light sources within the range of 390 to 530 nm results from the activity of cryptochrome with no other photoreceptor having a significant primary role at the fluence range tested. In both green- and norflurazon-treated (chlorophyll-deficient) seedlings, cryptochrome activity is fairly uniform throughout its range of maximal response (390-480 nm), with no sharply defined peak at 450 nm; however, activity at longer wavelengths was disproportionately enhanced in CRY1-overexpressing seedlings as compared with wild type. The action spectrum does not correlate well with the absorption spectra either of purified recombinant cryptochrome photoreceptor or to that of a second class of blue-light photoreceptor, phototropin (PHOT1 and PHOT2). Photoreceptor concentration as determined by western-blot analysis showed a greater stability of CRY2 protein under the monochromatic light conditions used in this study as compared with broad band blue light, suggesting a complex mechanism of photoreceptor activation. The possible role of additional photoreceptors (in particular phytochrome A) in cryptochrome responses is discussed.     

向光素调节植物向光性及其与光敏色素/隐花色素的相互关系

蓝光受体向光素(PHOT1/PHOT2)调节蓝光诱导的植物运动反应, 包括植物向光性、叶绿体运动、气孔运动和叶片伸展等。其中, 向光素介导的植物向光性能够促使植物弯向光源, 确保其以最佳取向捕获光源, 优化光合作用。光敏色素和隐花色素作为光受体也参与植物的向光性调节。该文综述了向光素介导的拟南芥(Arabidopsis thaliana)下胚轴向光弯曲信号转导及其与光敏色素、隐花色素协同作用的分子机制, 以期为改造植物光捕获能力及提高光利用效率提供理论基础。     

Blue light-dependent in vivo and in vitro phosphorylation of Arabidopsis cryptochrome 1

Cryptochromes are photolyase-like blue/UV-A light receptors that regulate various light responses in animals and plants. Arabidopsis cryptochrome 1 (cry1) is the major photoreceptor mediating blue light inhibition of hypocotyl elongation. The initial photochemistry underlying cryptochrome function and regulation remain poorly understood. We report here a study of the blue light-dependent phosphorylation of Arabidopsis cry1. Cry1 is detected primarily as unphosphorylated protein in etiolated seedlings, but it is phosphorylated in plants exposed to blue light. Cry1 phosphorylation increases in response to increased fluence of blue light, whereas the phosphorylated cry1 disappears rapidly when plants are transferred from light to dark. Light-dependent cry1 phosphorylation appears specific to blue light, because little cry1 phosphorylation is detected in seedlings treated with red light or far-red light, and it is largely independent from phytochrome actions, because no phytochrome mutants tested significantly affect cry1 phosphorylation. The Arabidopsis cry1 protein expressed and purified from insect cells is phosphorylated in vitro in a blue light-dependent manner, consistent with cry1 undergoing autophosphorylation. To determine whether cry1 phosphorylation is associated with its function or regulation, we isolated and characterized missense cry1 mutants that express full-length CRY1 apoprotein. Mutant residues are found throughout the CRY1 coding sequence, but none of these inactive cry1 mutant proteins shows blue light-induced phosphorylation. These results demonstrate that blue light-dependent cry1 phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of cry1 is critical to its phosphorylation.     

Pathogen-associated molecular pattern recognition rather than development of tissue necrosis contributes to bacterial induction of systemic acquired resistance in Arabidopsis

Systemic acquired resistance (SAR) is usually described as a phenomenon whereby localized inoculation with a necrotizing pathogen renders a plant more resistant to subsequent pathogen infection. Here we show that Pseudomonas syringae strains for which Arabidopsis thaliana represents a non-host plant systemically elevate resistance although the underlying interactions neither trigger a hypersensitive response nor cause necrotic disease symptoms. A similar enhancement of systemic resistance was observed when elicitor-active preparations of two typical bacterial pathogen-associated molecular patterns (PAMPs), flagellin and lipopolysaccharides (LPS), were applied in a localized manner. Several lines of evidence indicate that the observed systemic resistance responses are identical to SAR. Localized applications of non-adapted bacteria, flagellin or LPS elevate levels of the SAR regulatory metabolite salicylic acid (SA) and pathogenesis-related (PR) gene expression not only in treated but also in distant leaves. All treatments also systemically increase expression of the SAR marker gene FLAVIN-DEPENDENT MONOOXYGENASE 1. Further, a whole set of SAR-deficient Arabidopsis lines, including mutants in SA biosynthesis and signalling, are impaired in establishing the systemic resistance response triggered by non-host bacteria or PAMPs. We also show that the magnitude of defence reactions such as SA accumulation, PR gene expression or camalexin accumulation induced at sites of virulent or avirulent P. syringae inoculation but not the extent of tissue necrosis during these interactions determines the extent of SAR in distant leaves. Our data indicate that PAMPs significantly contribute to SAR initiation in Arabidopsis and that tissue necroses at inoculation sites are dispensable for SAR activation.     

Functional interaction of cryptochrome 1 and phytochrome D

Arabidopsis thaliana wild-type and single, double and triple mutants lacking phytochrome A (phyA-201), phytochrome B (phyB-5), phytochrome D (phyD-1), phytochrome E (phyE-1), cryptochrome 1 (hy4-2.23n) and cryptochrome 2 (fha-1) were used to study the photoreceptor signal-transduction network. The inhibition of hypocotyl elongation was analysed using pulses of red light preceded by a pre-irradiation of white light. The interactions of phyA, phyB and cry1 have been studied in a series of previous papers. Here we focus on the signal transduction initiated by phyD. We observed that phyD can partly substitute for the loss of phyB. Specifically, in the phyB background, red pulses were only effective if both cry1 and phyD were present. The response to red pulses, enabled by the pre-irradiation of white light, was completely reversible by far-red light. Loss of reversibility occurred with an apparent half-life of 2 h, similar to the half-life of 3 h observed for the effect mediated by phyB. Furthermore, we could show that the response to an end-of-day far-red pulse in phyB depends on both phyD and cry1. In contrast to phyD, a functional interaction of phyE and cry1 could not be detected in Arabidopsis seedlings.     

Hierarchical coupling of phytochromes and cryptochromes reconciles stability and light modulation of Arabidopsis development

In plants, development is a continuing process that takes place under strong fluctuations of the light environment. Here we show that in Arabidopsis thaliana plants grown under intense white light, coupling of the photoreceptor cryptochrome 2 to developmental processes is broader than previously appreciated. Compared to the wild type, the cry2 mutant showed reduced activity of a Lhcb1*2 promoter fused to a reporter, and delayed flowering. The cry2 mutation also reduced the inhibition of hypocotyl growth, the unfolding of the cotyledons, the rate of leaf production during the vegetative phase, and the pace of development after transition to the reproductive stage; but these effects were obvious only in the absence of cryptochrome 1 and in some cases phytochrome A and/or phytochrome B. Complementary, the cry2 mutation uncovered novel roles for cryptochrome 1 and phytochrome A. The activity of the Lhcb1*2 promoter was higher in the cry1 cry2 mutant than in the cry2 mutant, suggesting that cry1 could be involved in blue-light repression of photosynthetic genes. Surprisingly, the phyA cry1 cry2 triple mutant flowered earlier and showed better response to photoperiod than the cry1 cry2 double mutant, indicating that phyA is involved in light repression of flowering. Growth and development were severely impaired in the quadruple phyA phyB cry1 cry2 mutant. We propose that stability and light modulation of development are achieved by simultaneous coupling of phytochrome A, phytochrome B, cryptochrome 1 and cryptochrome 2 to developmental processes, in combination with context-dependent hierarchy of their relative activities.     

Degradation of Arabidopsis CRY2 is regulated by SPA proteins and phytochrome A

The UV-A/blue light photoreceptor crytochrome2 (cry2) plays a fundamental role in the transition from the vegetative to the reproductive phase in the facultative long-day plant Arabidopsis thaliana. The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome. COP1 is involved in cry2 degradation, but several cop1 mutants show only reduced but not abolished cry2 degradation. SUPPRESSOR OF PHYA-105 (SPA) proteins are known to work in concert with COP1, and recently direct physical interaction between cry2 and SPA1 was demonstrated. Thus, we hypothesized that SPA proteins could also play a role in cry2 degradation. To this end, we analyzed cry2 protein levels in spa mutants. In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner. Consistent with a role of SPA proteins in phytochrome A (phyA) signaling, a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells. Our results suggest that cry2 stability is controlled by SPA and phyA, thus providing more information on the molecular mechanisms of interaction between cryptochrome and phytochrome photoreceptors.     

Phototropins but not cryptochromes mediate the blue light-specific promotion of stomatal conductance, while both enhance photosynthesis and transpiration under full sunlight

Leaf epidermal peels of Arabidopsis (Arabidopsis thaliana) mutants lacking either phototropins 1 and 2 (phot1 and phot2) or cryptochromes 1 and 2 (cry1 and cry2) exposed to a background of red light show severely impaired stomatal opening responses to blue light. Since phot and cry are UV-A/blue light photoreceptors, they may be involved in the perception of the blue light-specific signal that induces the aperture of the stomatal pores. In leaf epidermal peels, the blue light-specific effect saturates at low irradiances; therefore, it is considered to operate mainly under the low irradiance of dawn, dusk, or deep canopies. Conversely, we show that both phot1 phot2 and cry1 cry2 have reduced stomatal conductance, transpiration, and photosynthesis, particularly under the high irradiance of full sunlight at midday. These mutants show compromised responses of stomatal conductance to irradiance. However, the effects of phot and cry on photosynthesis were largely nonstomatic. While the stomatal conductance phenotype of phot1 phot2 was blue light specific, cry1 cry2 showed reduced stomatal conductance not only in response to blue light, but also in response to red light. The levels of abscisic acid were elevated in cry1 cry2. We conclude that considering their effects at high irradiances cry and phot are critical for the control of transpiration and photosynthesis rates in the field. The effects of cry on stomatal conductance are largely indirect and involve the control of abscisic acid levels.     

Arabidopsis enhanced disease susceptibility mutants exhibit enhanced susceptibility to several bacterial pathogens and alterations in PR-1 gene expression.

To identify plant defense responses that limit pathogen attack, Arabidopsis eds mutants that exhibit enhanced disease susceptibility to the virulent bacterial pathogen Pseudomonas syringae pv maculicola ES4326 were previously identified. In this study, we show that each of four eds mutants (eds5-1, eds6-1, eds7-1, and eds9-1) has a distinguishable phenotype with respect to the degree of susceptibility to a panel of bacterial phytopathogens and the ability to activate pathogenesis-related PR-1 gene expression after pathogen attack. None of the four eds mutants exhibited observable defects in mounting a hypersensitive response. Although all four eds mutants were also capable of mounting a systemic acquired resistance response, enhanced growth of P. s. maculicola ES4326 was still apparent in the secondarily infected leaves of three of the eds mutants. These data indicate that eds genes define a diverse set of previously unknown defense responses that affect resistance to virulent pathogens.     

Temperature-dependent internode elongation in vegetative plants of Arabidopsis thaliana lacking phytochrome B and cryptochrome 1

 Vegetative plants of Arabidopsis thaliana (L.) Heynh. form a compact rosette of leaves in which internode growth is virtually arrested. Rapid extension of the internodes occurs after flower buds are present in the reproductive apex. Under natural radiation, continuous light from fluorescent lamps, or short photoperiods of light from fluorescent lamps, plants of the phyB cry1 double mutant (lacking both phytochrome B and cryptochrome 1) did not form normal rosettes because all the internodes showed some degree of elongation. Internode elongation was weak in the phyB single mutant and absent in the cry1 mutant, indicating redundancy between phytochrome B and cryptochrome 1. The absence of phytochrome A caused no effects. The failure to form normal rosettes was conditional because internode elongation was arrested at low temperatures in all the mutant combinations. In contrast, the temperature dependence of phytochrome B and cryptochrome 1 effects on hypocotyl growth was weak. The elongation of the internodes in phyB cry1 was not accompanied by early flowering as showed by the lack of effects on the final number of leaves. Apex dissection indicated that in phyB cry1 double mutants internode elongation anticipated the transition from the vegetative to the reproductive stage. Thus, stem growth in Arabidopsis thaliana is not fully dependent on the program of reproductive development. Received: 2 June 1999 / Accepted: 13 August 1999     

Intramolecular co-action of two independent photosensory modules in the fern phytochrome 3

Fern phytochrome3/neochrome1 (phy3/neo1) is a chimeric photoreceptor composed of a phytochrome-chromophore binding domain and an almost full-length phototropin. phy3 thus contains two different light-sensing modules; a red/far-red light receptor phytochrome and a blue light receptor phototropin. phy3 induces both red light- and blue light-dependent phototropism in phototropin-deficient Arabidopsis thaliana (phot1 phot2) seedlings. The red-light response is dependent on the phytochrome module of phy3, and the blue-light response is dependent on the phototropin module. We recently showed that both the phototropin-sensing module and the phytochrome-sensing module mediate the blue light-dependent phototropic response. Particularly under low-light conditions, these two light-sensing modules cooperate to induce the blue light-dependent phototropic response. This intramolecular co-action of two independent light-sensing modules in phy3 enhances light sensitivity, and perhaps allowed ferns to adapt to the low-light canopy conditions present in angiosperm forests.