Root-localized phytochrome chromophore synthesis is required for photoregulation of root elongation and impacts root sensitivity to jasmonic acid in Arabidopsis

Plants exhibit organ- and tissue-specific light responses. To explore the molecular basis of spatial-specific phytochrome-regulated responses, a transgenic approach for regulating the synthesis and accumulation of the phytochrome chromophore phytochromobilin (PΦB) was employed. In prior experiments, transgenic expression of the BILIVERDIN REDUCTASE (BVR) gene was used to metabolically inactivate biliverdin IXα, a key precursor in the biosynthesis of PΦB, and thereby render cells accumulating BVR phytochrome deficient. Here, we report analyses of transgenic Arabidopsis (Arabidopsis thaliana) lines with distinct patterns of BVR accumulation dependent upon constitutive or tissue-specific, promoter-driven BVR expression that have resulted in insights on a correlation between root-localized BVR accumulation and photoregulation of root elongation. Plants with BVR accumulation in roots and a PΦB-deficient elongated hypocotyl2 (hy2-1) mutant exhibit roots that are longer than those of wild-type plants under white illumination. Additional analyses of a line with root-specific BVR accumulation generated using a GAL4-dependent bipartite enhancer-trap system confirmed that PΦB or phytochromes localized in roots directly impact light-dependent root elongation under white, blue, and red illumination. Additionally, roots of plants with constitutive plastid-localized or root-specific cytosolic BVR accumulation, as well as phytochrome chromophore-deficient hy1-1 and hy2-1 mutants, exhibit reduced sensitivity to the plant hormone jasmonic acid (JA) in JA-dependent root inhibition assays, similar to the response observed for the JA-insensitive mutants jar1 and myc2. Our analyses of lines with root-localized phytochrome deficiency or root-specific phytochrome depletion have provided novel insights into the roles of root-specific PΦB, or phytochromes themselves, in the photoregulation of root development and root sensitivity to JA.     

Spatial-specific regulation of root development by phytochromes in Arabidopsis thaliana

Distinct tissues and organs of plants exhibit dissimilar responses to light exposure – cotyledon growth is promoted by light, whereas hypocotyl growth is inhibited by light. Light can have different impacts on root development, including impacting root elongation, morphology, lateral root proliferation and root tropisms. In many cases, light inhibits root elongation. There has been much attention given to whether roots themselves are the sites of photoperception for light that impacts light-dependent growth and development of roots. A number of approaches including photoreceptor localization in planta, localized irradiation and exposure of dissected roots to light have been used to explore the site(s) of light perception for the photoregulation of root development. Such approaches have led to the observation that photoreceptors are localized to roots in many plant species, and that roots are capable of light absorption that can alter morphology and/or gene expression. Our recent results show that localized depletion of phytochrome photoreceptors in Arabidopsis thaliana disrupts root development and root responsiveness to the plant hormone jasmonic acid. Thus, root-localized light perception appears central to organ-specific, photoregulation of growth and development in roots.     

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.     

Photoresponses of transgenic Arabidopsis seedlings expressing introduced phytochrome B-encoding cDNAs: evidence that phytochrome A and phytochrome B have distinct photoregulatory functions

The photocontrol of hypocotyl elongation has been studied in two transgenic lines of Arabidopsis thaliana which contain elevated levels of phytochrome B encoded by either an introduced rice- or Arabidopsis -derived cDNA driven by the 35S CaMV promoter. Inhibition of hypocotyl growth in etiolated seedlings of the phyB -transformed lines was saturated at photon fluence rates of continuous red light (R) which were markedly lower than those required for inhibition of growth in seedlings of the isogenic wild-type (WT). Inhibition of hypocotyl growth in etiolated seedlings of the phyB -transgenic lines under continuous far-red irradiation (FR), however, showed the same relationship with fluence rate as WT. Light-grown seedlings of the phyB -transgenic lines responded to end-of-day FR by an acceleration of growth, in a manner comparable with WT. This response was unaltered when the end-of-day FR was extended from a 15 min pulse to 14 h of continuous irradiation. The response of light-grown, phyB -transformed seedlings to decreasing R:FR ratio was also qualitatively similar to WT, i.e. increased elongation growth of the hypocotyl and petioles occurred under low R:FR quantum ratio. However, absolute elongation growth was markedly less in the transgenic seedlings at all R:FR ratios tested than in WT. Together, these data indicate that seedlings over-expressing phytochrome B are more responsive to R than are WT, but are unaltered in their responsiveness to FR. By contrast, seedlings overexpressing phytochrome A are more responsive than WT to both R and FR; whereas the phytochrome B-deficient mutant hy3 is unresponsive to R while retaining WT-like responsiveness to FR. These data indicate that in WT etiolated seedlings phytochrome A mediates the effects of continuous FR, and phytochrome B the effects of continuous R. The evidence thus supports the conclusion that these two molecular species of the photoreceptor have differential regulatory roles in the plant.     

Selected Components of the Shade-Avoidance Syndrome Are Displayed in a Normal Manner in Mutants of Arabidopsis thaliana and Brassica rapa Deficient in Phytochrome B

Several growth parameters associated with the phytochrome-mediated shade avoidance syndrome have been measured in seedlings and mature plants of a wild-type and a hy3 mutant of Arabidopsis thaliana deficient in phytochrome B. Growth parameters were compared in plants grown in either white light (high red:far-red [R:FR] ratio) or white light plus added far-red (FR) light (low R:FR ratio). Wild-type Arabidopsis exhibited increased hypocotyl and petiole extension under a low, compared with a high, R:FR ratio. The hy3 mutant did not respond to low R:FR ratio by increase in hypocotyl or petiole length. Extension growth of wild-type plants was stimulated by brief end-of-day FR pulses, but similar treatment had no effect on extension growth of hy3 mutant plants. However, some responses to low R:FR ratio seen in the wild-type plants were also evident in the hy3 mutants. The number of days to bolting, the developmental stage at bolting, the leaf area, and the specific stem weight (weight per unit of length) all decreased in the wild-type and hy3 seedlings in response to low R:FR ratio. Low R:FR ratio caused a larger decrease in leaf area and specific stem weight in the mutant seedlings than in wild-type seedlings. The effects of low R:FR ratio on leaf area and specific stem weight were opposite to those of the hy3 lesion, which resulted in increased leaf area and specific stem weight in comparison with the wild type. Both leaf area and specific stem weight responses to low R:FR ratio also were unchanged in the ein mutant of Brassica rapa, known to be deficient in phytochrome B. These responses represent components of the shade-avoidance syndrome, and, consequently, the results indicate that phytochrome B cannot be solely responsible for the perception of R:FR ratio and the induction of shade-avoidance responses. The hypothesis is proposed that different phytochromes may be responsible for the regulation of extension growth and the regulation of lateral or radial expansion.     

FIN5 positively regulates far-red light responses in Arabidopsis thaliana

We report the characterization of a semi-dominant mutation fin5-1 (far-red insensitive 5-1) of Arabidopsis, which was isolated from genetic screening of phytochrome A (phyA) signaling components. Plants with the fin5-1 mutation exhibited a long hypocotyl phenotype when grown under far-red (FR) light, but not under red light. Physiological analyses implied that FIN5 might be differentially involved in diverse responses that are regulated by phyA under continuous FR light. Anthocyanin accumulation, gravitropic response of hypocotyl growth, and FR light-preconditioned blocking of greening were also impaired in the fin5-1 mutant, whereas photoperiodic floral induction was not, if at all, significantly affected. Moreover, light-regulated expression of the CHS, PORA and PsbS genes was attenuated in fin5-1 mutant plants, while the light-induced expression of CAB was normal. The mutation exhibited semi-dominance regarding control of hypocotyl growth in FR light. We suggest that FIN5 defines a novel branch in the network of phyA signaling in Arabidopsis.     

Phytochrome E Controls Light-Induced Germination of Arabidopsis

Germination of Arabidopsis seeds is light dependent and under phytochrome control. Previously, phytochromes A and B and at least one additional, unspecified phytochrome were shown to be involved in this process. Here, we used a set of photoreceptor mutants to test whether phytochrome D and/or phytochrome E can control germination of Arabidopsis. The results show that only phytochromes B and E, but not phytochrome D, participate directly in red/far-red light (FR)-reversible germination. Unlike phytochromes B and D, phytochrome E did not inhibit phytochrome A-mediated germination. Surprisingly, phytochrome E was required for germination of Arabidopsis seeds in continuous FR. However, inhibition of hypocotyl elongation by FR, induction of cotyledon unfolding, and induction of agravitropic growth were not affected by loss of phytochrome E. Therefore, phytochrome E is not required per se for phytochrome A-mediated very low fluence responses and the high irradiance response. Immunoblotting revealed that the need of phytochrome E for germination in FR was not caused by altered phytochrome A levels. These results uncover a novel role of phytochrome E in plant development and demonstrate the considerable functional diversification of the closely related phytochromes B, D, and E.     

Arabidopsis thaliana TERMINAL FLOWER2 is involved in light-controlled signalling during seedling photomorphogenesis

Plants respond to changes in the environment by altering their growth pattern. Light is one of the most important environmental cues and affects plants throughout the life cycle. It is perceived by photoreceptors such as phytochromes that absorb light of red and far-red wavelengths and control, for example, seedling de-etiolation, chlorophyll biosynthesis and shade avoidance response. We report that the terminal flower2 (tfl2) mutant, carrying a mutation in the Arabidopsis thaliana HETEROCHROMATIN PROTEIN1 homolog, functions in negative regulation of phytochrome dependent light signalling. tfl2 shows defects in both hypocotyl elongation and shade avoidance response. Double mutant analysis indicates that mutants of the red/far-red light absorbing phytochrome family of plant photoreceptors, phyA and phyB, are epistatic to tfl2 in far-red and red light, respectively. An overlap between genes regulated by light and by auxin has earlier been reported and, in tfl2 plants light-dependent auxin-regulated genes are misexpressed. Further, we show that TFL2 binds to IAA5 and IAA19 suggesting that TFL2 might be involved in regulation of phytochrome-mediated light responses through auxin action.     

Genetic separation of phototropism and blue light inhibition of stem elongation

Blue light-induced regulation of cell elongation is a component of the signal response pathway for both phototropic curvature and inhibition of stem elongation in higher plants. To determine if blue light regulates cell elongation in these responses through shared or discrete pathways, phototropism and hypocotyl elongation were investigated in several blue light response mutants in Arabidopsis thaliana. Specifically, the blu mutants that lack blue light-dependent inhibition of hypocotyl elongation were found to exhibit a normal phototropic response. In contrast, a phototropic null mutant (JK218) and a mutant that has a 20- to 30-fold shift in the fluence dependence for first positive phototropism (JK224) showed normal inhibition of hypocotyl elongation in blue light. F1 progeny of crosses between the blu mutants and JK218 showed normal phototropism and inhibition of hypocotyl elongation, and approximately 1 in 16 F2 progeny were double mutants lacking both responses. Thus, blue light-dependent inhibition of hypocotyl elongation and phototropism operate through at least some genetically distinct components.     

Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light

A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162–170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.Abbreviations FR far-red light - R red light - WL white light - WL + FR white light supplemented with FR - HIR high-irradiance response - PAR photosynthetically active radiation - Pr, Pfr R- and FR-absorbing forms of phytochrome - Ptot total phytochrome - phyA (PhyA) gene (encoded protein) for phytochrome - WT wild type This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.M.; J.R. Cherry and R.D. Vierstra, (Department of Horticulture, University of Wisconsin-Madison, USA) are thanked for the provision of the transgenic tobacco line.     

Phytochrome-mediated phototropism in de-etiolated seedlings : occurrence and ecological significance

Phototropic responses to broadband far red (FR) radiation were investigated in fully de-etiolated seedlings of a long-hypocotyl mutant (lh) of cucumber (Cucumis sativus L.), which is deficient in phytochrome-B, and its near isogenic wild type (WT). Continuous unilateral FR light provided against a background of white light induced negative curvatures (i.e. bending away from the FR light source) in hypocotyls of WT seedlings. This response was fluence-rate dependent and was absent in the lh mutant, even at very high fluence rates of FR. The phototropic effect of FR light on WT seedlings was triggered in the hypocotyls and occurred over a range of fluence rates in which FR was very effective in promoting hypocotyl elongation. FR light had no effect on elongation of lh-mutant hypocotyls. Seedlings grown in the field showed negative phototropic responses to the proximity of neighboring plants that absorbed blue (B) and red light and back-reflected FR radiation. The bending response was significantly larger in WT than in lh seedlings. Responses of WT and lh seedlings to lateral B light were very similar; however, elimination of the lateral B light gradients created by the proximity of plant neighbors abolished the negative curvature only in the case of lh seedlings. More than 40% of the total hypocotyl curvature induced in WT seedlings by the presence of neighboring plants was present after equilibrating the fluence rates of B light received by opposite sides of the hypocotyl. These results suggest that: (a) phytochrome functions as a phototropic sensor in de-etiolated plants, and (b) in patchy canopy environments, young seedlings actively project new leaves into light gaps via stem bending responses elicited by the B-absorbing photoreceptor(s) and phytochrome.     

Suppressors of an Arabidopsis thaliana phyB mutation identify genes that control light signaling and hypocotyl elongation.

Ambient light controls the development and physiology of plants. The Arabidopsis thaliana photoreceptor phytochrome B (PHYB) regulates developmental light responses at both seedling and adult stages. To identify genes that mediate control of development by light, we screened for suppressors of the long hypocotyl phenotype caused by a phyB mutation. Genetic analyses show that the shy (short hypocotyl) mutations we have isolated fall in several loci. Phenotypes of the mutants suggest that some of the genes identified have functions in control of light responses. Other loci specifically affect cell elongation or expansion.     

Flowering responses to altered expression of phytochrome in mutants and transgenic lines of Arabidopsis thaliana (L.) Heynh.

The long-day plant Arabidopsis thaliana (L.) Heynh. flowers early in response to brief end-of-day (EOD) exposures to far-red light (FR) following a fluorescent short day of 8 h. FR promotion of flowering was nullified by subsequent brief red light (R) EOD exposure, indicating phytochrome involvement. The EOD response to R or FR is a robust measure of phytochrome action. Along with their wild-type (WT) parents, mutants deficient in either phytochrome A or B responded similarly to the EOD treatments. Thus, neither phytochrome A nor B exclusively regulated flowering, although phytochrome B controlled hypocotyl elongation. Perhaps a third phytochrome species is important for the EOD responses of the mutants and/or their flowering is regulated by the amount of the FR-absorbing form of phytochrome, irrespective of the phytochrome species. Overexpression of phytochrome A or phytochrome B resulted in differing photoperiod and EOD responses among the genotypes. The day-neutral overexpressor of phytochrome A had an EOD response similar to all of the mutants and WTs, whereas R EOD exposure promoted flowering in the overexpressor of phytochrome B and FR EOD exposure inhibited this promotion. The comparisons between relative flowering times and leaf numbers at flowering of the over-expressors and their WTs were not consistent across photoperiods and light treatments, although both phytochromes A and B contributed to regulating flowering of the transgenic plants.     

Photoperiodism and photocontrol of stem elongation in two photomorphogenic mutants of Pisum sativum L.

The photomorphogenic mutation lv in the garden pea (Pisum sativum L.), which appears to reduce the response to light-stable phytochrome, has been isolated on a tall, late photoperiodic genetic background and its effects further characterised. Plants possessing lv have a reduced flowering response to photoperiod relative to wild-type plants, indicating that light-stable phytochrome may have a flower-inhibitory role in the flowering response of long-day plants to photoperiod. In general, lv plants are longer and have reduced leaf development relative to Lv plants. These differences are maximised under continuous light from fluorescent lamps (containing negligible far-red (FR) light), and decrease with addition of FR to the incident light. Enrichment of white light from fluorescent lamps with FR promotes stem elongation in the wild type but causes a reduction in elongation in the lv mutant. This “negative” shade-avoidance response appears to be the consequence of a strong inhibitory effect of light rich in FR, revealed in lv plants in the absence of a normal response to red (R) light. These results indicate that the wild-type response to the R: FR ratio may be comprised of two distinct photoresponses, one in which FR supplementation promotes elongation by reducing the inhibitory effect of R, and the other in which light rich in FR actively inhibits elongation. This hypothesis is discussed in relation to functional differentiation of phytochrome types in the light-grown plant. Gene lw has been reported previously to reduce internode length and the response to gibberellin A₁, and to delay flowering. The present study shows that the lw mutation confers an increased response to photoperiod. In all these responses the lw phenotype is superficially “opposite” to the lv phenotype. The possibility that the mutation might primarily affect light perception was therefore considered. The degree of dwarfing of lw plants was found to depend upon light quality and quantity. Dwarfing is more extreme in plants grown under continuous R light than in those grown in continuous FR or blue light or in darkness. Studies of the fluence-rate response show that the lw mutation imparts a lower fluence requirement for inhibition of elongation by white light from fluorescent lamps. Dark-grown lw plants are more strongly inhibited by a R pulse than are wild-type plants but, as in the wild type, this inhibition remains reversible by FR. Light-grown lw plants show an exaggerated elongation response to end-of-day FR light. Taken together, these findings indicate that the lw mutant may be hypersensitive to phytochrome action.     

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.     

Gibberellins control Arabidopsis hypocotyl growth via regulation of cellular elongation

The gibberellins (GAs) are endogenous regulators of plant growth. Experiments are described here that test the hypothesis that GA regulates hypocotyl growth by altering the extent of hypocotyl cell elongation. These experiments use GA-deficient and altered GA-response mutants of Arabidopsis thaliana (L.) Heyhn. It is shown that GA regulates elongation, in both light- and dark-grown hypocotyls, by influencing the rate and final extent of cellular elongation. However, light- and dark-grown hypocotyls exhibit markedly different GA dose-response relationships. The length of dark-grown hypocotyls is relatively unaffected by exogenous GA, whilst light-grown hypocotyl length is significantly increased by exogenous GA. Further analysis suggests that GA control of hypocotyl length is close to saturation in dark-grown hypocotyls, but not in light grown hypocotyls. The results show that a large range of possible hypocotyl lengths is achieved via dose-dependent GA-regulated alterations in the degree of elongation of individual hypocotyl cells.Key words: Arabidopsis, cell elongation, gibberellin (GA), GA mutants, hypocotyl.