Phytochrome-mediated agravitropism in Arabidopsis hypocotyls requires GIL1 and confers a fitness advantage

Plants use specialized photoreceptors to detect the amount, quality, periodicity and direction of light and to modulate their growth and development accordingly. These regulatory light signals often interact with other environmental cues. Exposure of etiolated Arabidopsis seedlings to red (R) or far-red (FR) light causes hypocotyls to grow in random orientations with respect to the gravitational vector, thus overcoming the signal from gravity to grow upwards. This light response, mediated by either phytochrome A or phytochrome B, represents a prime example of cross-talk between environmental signalling systems. Here, we report the isolation the mutant gil1 (for gravitropic in the light) in which hypocotyls continue to grow upwards after exposure of seedlings to R or FR light. The gil1 mutant displays no other phenotypic alterations in response to gravity or light. Cloning of GIL1 has identified a novel gene that is necessary for light-dependent randomization of hypocotyl growth orientation. Using gil1, we have demonstrated that phytochrome-mediated randomization of Arabidopsis hypocotyl orientation provides a fitness advantage to seedlings developing in patchy, low-light environments.     

PHYTOCHROME KINASE SUBSTRATE1 regulates root phototropism and gravitropism

Light promotes the expression of PHYTOCHROME KINASE SUBSTRATE1 (PKS1) in the root of Arabidopsis thaliana, but the function of PKS1 in this organ is unknown. Unilateral blue light induced a negative root phototropic response mediated by phototropin 1 in wild-type seedlings. This response was absent in pks1 mutants. In the wild type, unilateral blue light enhanced PKS1 expression in the subapical region of the root several hours before bending was detectable. The negative phototropism and the enhanced PKS1 expression in response to blue light required phytochrome A (phyA). In addition, the pks1 mutation enhanced the root gravitropic response when vertically oriented seedlings were placed horizontally. The negative regulation of gravitropism by PKS1 occurred even in dark-grown seedlings and did not require phyA. Blue light also failed to induce negative phototropism in pks1 under reduced gravitational stimulation, indicating that the effect of pks1 on phototropism is not simply the consequence of the counteracting effect of enhanced gravitropism. We propose a model where the background level of PKS1 reduces gravitropism. After a phyA-dependent increase in its expression, PKS1 positively affects root phototropism and both effects contribute to negative curvature in response to unilateral blue light.     

Hypocotyl growth orientation in blue light is determined by phytochrome A inhibition of gravitropism and phototropin promotion of phototropism

How developing seedlings integrate gravitropic and phototropic stimuli to determine their direction of growth is poorly understood. In this study we tested whether blue light influences hypocotyl gravitropism in Arabidopsis. Phototropin1 (phot1) triggers phototropism under low fluence rates of blue light but, at least in the dark, has no effect on gravitropism. By analyzing the growth orientation of phototropism-deficient seedlings in response to gravitropic and phototropic stimulations we show that blue light not only triggers phototropism but also represses hypocotyl gravitropism. At low fluence rates of blue light phot1 mutants were agravitropic. In contrast, phyAphot1 double mutants grew exclusively according to gravity demonstrating that phytochrome A (phyA) is necessary to inhibit gravitropism. Analyses of phot1cry1cry2 triple mutants indicate that cryptochromes play a minor role in this response. Thus the optimal growth orientation of hypocotyls is determined by the action of phyA-suppressing gravitropism and the phototropin-triggering phototropism. It has long been known that phytochromes promote phototropism but the mechanism involved is still unknown. Our data show that by inhibiting gravitropism phyA acts as a positive regulator of phototropism.     

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.     

RCN1-regulated phosphatase activity and EIN2 modulate hypocotyl gravitropism by a mechanism that does not require ethylene signaling

The roots curl in naphthylphthalamic acid1 (rcn1) mutant of Arabidopsis (Arabidopsis thaliana) has altered auxin transport, gravitropism, and ethylene response, providing an opportunity to analyze the interplay between ethylene and auxin in control of seedling growth. Roots of rcn1 seedlings were previously shown to have altered auxin transport, growth, and gravitropism, while rcn1 hypocotyl elongation exhibited enhanced ethylene response. We have characterized auxin transport and gravitropism phenotypes of rcn1 hypocotyls and have explored the roles of auxin and ethylene in controlling these phenotypes. As in roots, auxin transport is increased in etiolated rcn1 hypocotyls. Hypocotyl gravity response is accelerated, although overall elongation is reduced, in etiolated rcn1 hypocotyls. Etiolated, but not light grown, rcn1 seedlings also overproduce ethylene, and mutations conferring ethylene insensitivity restore normal hypocotyl elongation to rcn1. Auxin transport is unaffected by treatment with the ethylene precursor 1-aminocyclopropane carboxylic acid in etiolated hypocotyls of wild-type and rcn1 seedlings. Surprisingly, the ethylene insensitive2-1 (ein2-1) and ein2-5 mutations dramatically reduce gravitropic bending in hypocotyls. However, the ethylene resistant1-3 (etr1-3) mutation does not significantly affect hypocotyl gravity response. Furthermore, neither the etr1 nor the ein2 mutation abrogates the accelerated gravitropism observed in rcn1 hypocotyls, indicating that both wild-type gravity response and enhanced gravity response in rcn1 do not require an intact ethylene-signaling pathway. We therefore conclude that the RCN1 protein affects overall hypocotyl elongation via negative regulation of ethylene synthesis in etiolated seedlings, and that RCN1 and EIN2 modulate hypocotyl gravitropism and ethylene responses through independent pathways.     

The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway

The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.     

Reduced phototropism in pks mutants may be due to altered auxin‐regulated gene expression or reduced lateral auxin transport

Phototropism allows plants to orient their photosynthetic organs towards the light. In Arabidopsis, phototropins 1 and 2 sense directional blue light such that phot1 triggers phototropism in response to low fluence rates, while both phot1 and phot2 mediate this response under higher light conditions. Phototropism results from asymmetric growth in the hypocotyl elongation zone that depends on an auxin gradient across the embryonic stem. How phototropin activation leads to this growth response is still poorly understood. Members of the phytochrome kinase substrate (PKS) family may act early in this pathway, because PKS1, PKS2 and PKS4 are needed for a normal phototropic response and they associate with phot1 in vivo. Here we show that PKS proteins are needed both for phot1‐ and phot2‐mediated phototropism. The phototropic response is conditioned by the developmental asymmetry of dicotyledonous seedlings, such that there is a faster growth reorientation when cotyledons face away from the light compared with seedlings whose cotyledons face the light. The molecular basis for this developmental effect on phototropism is unknown; here we show that PKS proteins play a role at the interface between development and phototropism. Moreover, we present evidence for a role of PKS genes in hypocotyl gravi‐reorientation that is independent of photoreceptors. pks mutants have normal levels of auxin and normal polar auxin transport, however they show altered expression patterns of auxin marker genes. This situation suggests that PKS proteins are involved in auxin signaling and/or lateral auxin redistribution.     

Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis

Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome–dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation.     

The hypocotyl chloroplast plays a role in phototropic bending of Arabidopsis seedlings: developmental and genetic evidence

Chloroplasts of guard cells and coleoptiles have been implicated in the sensory transduction of blue light. The present study was aimed at establishing whether the chloroplast of the hypocotyl from Arabidopsis, another blue light-responding organ, has similar characteristics to that of sensory-transducing guard cell and coleoptile chloroplasts. Results showed that the phototropic curvature and arch length induced by blue light in Arabidopsis seedlings matched the distribution of mature chloroplasts in the bending hypocotyl. The bending arch consistently included the region of the hypocotyl containing mature chloroplasts, and never extended beyond that region. Manipulation of the extent of greening of dark-grown hypocotyls by varying red light pretreatments elicited blue light-stimulated curvatures and arch lengths that depended on the duration of the red light pretreatment and on the distribution of mature chloroplasts in the hypocotyl. Albino psd2 mutants of Arabidopsis, which lack mature chloroplasts, are devoid of phototropic sensitivity under conditions in which wild-type seedlings show large curvatures. The star mutant of Arabidopsis has a delayed greening and a delayed phototropic response as compared with wild type. Measurements of photosynthetic oxygen evolution and carbon fixation, dark respiration, and light-dependent zeaxanthin formation in the hypocotyl showed features similar to those of guard cells and coleoptiles, and distinctly different from those of mesophyll tissue. These results indicate that the hypocotyl chloroplast has characteristics similar to those associated with guard cell and coleoptile chloroplasts, and that phototropic bending of Arabidopsis hypocotyls appears to require mature chloroplasts.     

PKS1 plays a role in red-light-based positive phototropism in roots

Aerial parts of plants curve towards the light (i.e. positive phototropism), and roots typically grow away from the light (i.e. negative phototropism). In addition, Arabidopsis roots exhibit positive phototropism relative to red light (RL), and this response is mediated by phytochromes A and B (phyA and phyB). Upon light stimulation, phyA and phyB interact with the phytochrome kinase substrate (PKS1) in the cytoplasm. In this study, we investigated the role of PKS1, along with phyA and phyB, in the positive phototropic responses to RL in roots. Using a high-resolution feedback system, we studied the phenotypic responses of roots of phyA, phyB, pks1, phyA pks1 and phyB pks1 null mutants as well as the PKS1-overexpressing line in response to RL. PKS1 emerged as an intermediary in the signalling pathways and appears to promote a negative curvature to RL in roots. In addition, phyA and phyB were both essential for a positive response to RL and act in a complementary fashion. However, either photoreceptor acting without the other results in negative curvature in response to red illumination so that the mode of action differs depending on whether phyA and phyB act independently or together. Our results suggest that PKS1 is part of a signalling pathway independent of phyA and phyB and that PKS1 modulates RL-based root phototropism.     

Nitric oxide regulates DELLA content and PIF expression to promote photomorphogenesis in Arabidopsis

The transition from etiolated to green seedlings involves a shift from hypocotyl growth-promoting conditions to growth restraint. These changes occur through a complex light-driven process involving multiple and tightly coordinated hormonal signaling pathways. Nitric oxide (NO) has been lately characterized as a regulator of plant development interacting with hormone signaling. Here, we show that Arabidopsis (Arabidopsis thaliana) NO-deficient mutant hypocotyls are longer than those from wild-type seedlings under red light but not under blue or far-red light. Accordingly, exogenous treatment with the NO donor sodium nitroprusside and mutant plants with increased endogenous NO levels resulted in reduced hypocotyl length. In addition to increased hypocotyl elongation, NO deficiency led to increased anthocyanin levels and reduced PHYB content under red light, all processes governed by phytochrome-interacting factors (PIFs). NO-deficient plants accordingly showed an enhanced expression of PIF3, PIF1, and PIF4. Moreover, exogenous NO increased the levels of the gibberellin (GA)-regulated DELLA proteins and shortened hypocotyls, likely through the negative regulation of the GA Insensitive Dwarf1 (GID1)-Sleepy1 (SLY1) module. Consequently, NO-deficient seedlings displayed up-regulation of SLY1, defective DELLA accumulation, and altered GA sensitivity, thus resulting in defective deetiolation under red light. Accumulation of NO in wild-type seedlings undergoing red light-triggered deetiolation and elevated levels of NO in the GA-deficient ga1-3 mutant in darkness suggest a mutual NO-GA antagonism in controlling photomorphogenesis. PHYB-dependent NO production promotes photomorphogenesis by a GID1-GA-SLY1-mediated mechanism based on the coordinated repression of growth-promoting PIF genes and the increase in the content of DELLA proteins.     

Genetic and transgenic evidence that phytochromes A and B act to modulate the gravitropic orientation of Arabidopsis thaliana hypocotyls

Hypocotyls of Arabidopsis thaliana exhibit negative gravitropism in the dark, growing against the gravity vector. The direction of growth is randomized in red light (R). In single mutants lacking either phytochrome A or B randomization of hypocotyl orientation in R is retained. However, a double mutant lacks this response, indicating that either phytochrome A or B is capable of inducing randomization and phytochrome A and B are the only phytochromes involved in this process. The induction of randomization was confirmed using lines that express to different levels PHYA and PHYB cDNAs. Overexpression of PHYA cDNAs induced randomization of hypocotyl orientation in the dark. Dark randomization was also seen in the phyB-1 mutant but not in two other phyB alleles, suggesting that dark randomization in the phyB-1 line may be due to a second mutation. When germination was induced by gibberellin, rather than exposure to brief white light, randomization in the dark associated with phytochrome A overproduction was not observed but was retained in the phyB-1 mutant. Overexpression of PHYB cDNAs induced a light-dependent randomization of hypocotyl orientation that responded to R:far-red light ratio. We conclude that the default situation in Arabidopsis hypocotyls is, therefore, negative gravitropism, and either phytochrome A or phytochrome B can mediate randomization.     

The Effects of Cytokinin and Light on Hypocotyl Elongation in Arabidopsis Seedlings Are Independent and Additive

Cytokinin has been reported to mimic some of the effects of light on de-etiolation responses in dark-grown Arabidopsis seedlings. The interaction between cytokinin and light was examined by analyzing cytokinin dose and light fluence effects on hypocotyl elongation in wild-type and mutant Arabidopsis seedlings with defects in light or hormone responses. It was found that (a) cytokinin and light-response systems have independent and additive effects on the inhibition of hypocotyl elongation and (b) either cytokinin or light can saturate the morphogenic responses. As a consequence, cytokinin has no effect on hypocotyl elongation under normal growth conditions because light levels saturate the hypocotyl inhibition response. To determine whether a functional light-response pathway is required for cytokinin responses, light-insensitive long hypocotyl (hy) mutants were tested for cytokinin responses. The hy mutants (hy1 to hy6) had normal cytokinin responses, except phyB-1 (hy3-1), in which hypocotyl elongation was insensitive to cytokinin. Cytokinin insensitivity in phyB-1 was attributed to an indirect effect of the mutation on cytokinin responses. The effects of cytokinin on the inhibition of hypocotyl elongation are largely mediated by ethylene, and blocking the ethylene-response pathway through the action of a cytokinin-resistant, ethylene-insensitive mutant (ckr1/ein2) had no effect on the light inhibition of hypocotyl elongation. These results do not support the idea that cytokinin mediates the action of light on hypocotyl elongation.     

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.      

A hormonal regulatory module that provides flexibility to tropic responses

Plants orient their growth depending on directional stimuli such as light and gravity, in a process known as tropic response. Tropisms result from asymmetrical accumulation of auxin across the responding organ relative to the direction of the stimulus, which causes differential growth rates on both sides of the organ. Here, we show that gibberellins (GAs) attenuate the gravitropic reorientation of stimulated hypocotyls of dark-grown Arabidopsis (Arabidopsis thaliana) seedlings. We show that the modulation occurs through induction of the expression of the negative regulator of auxin signaling INDOLE-3-ACETIC ACID INDUCIBLE19/MASSUGU2. The biological significance of this regulatory mechanism involving GAs and auxin seems to be the maintenance of a high degree of flexibility in tropic responses. This notion is further supported by observations that GA-deficient seedlings showed a much lower variance in the response to gravity compared to wild-type seedlings and that the attenuation of gravitropism by GAs resulted in an increased phototropic response. This suggests that the interplay between auxin and GAs may be particularly important for plant orientation under competing tropic stimuli.     

Involvement of an ABC transporter in a developmental pathway regulating hypocotyl cell elongation in the light

In the dark, plant seedlings follow the skotomorphogenetic developmental program, which results in hypocotyl cell elongation. When the seedlings are exposed to light, a switch to photomorphogenetic development occurs, and hypocotyl cell elongation is inhibited. We have manipulated the expression of the AtPGP1 (for Arabidopsis thaliana P glycoprotein1) gene in transgenic Arabidopsis plants by using sense and antisense constructs. We show that within a certain light fluence rate window, overexpression of the AtPGP1 gene under the control of the cauliflower mosaic virus 35S promoter causes plants to develop longer hypocotyls, whereas expression of the gene in antisense orientation results in hypocotyls shorter than those occurring in the wild type. In the dark, hypocotyls of transgenic and wild-type plants are indistinguishable. Because the AtPGP1 gene encodes a member of the superfamily of ATP binding cassette-containing (ABC) transporters, these results imply that a transport process is involved in a hypocotyl cell elongation pathway active in the light. The AtPGP1 transporter is localized in the plasmalemma, as indicated by immunohistochemical techniques and biochemical membrane separation methods. Analysis of the AtPGP1 expression pattern by using reporter gene constructs and in situ hybridization shows that in wild-type seedlings, AtPGP1 is expressed in both the root and shoot apices.     

Photoregulation of hypocotyl growth: geotropic evidence for the operation of two photosystems

Abstract. The rate of curvature of etiolated cress ( Lepi-dium sativum L. ) hypocotyls in response to gravity (negative geotropism) was retarded by red or blue light; far-red irradiation was without effect. The timing of the irradiation period in relation to the presentation for geostimulus markedly affected the response. When seedlings were irradiated during the 1–2 h period of geostimulus, blue light was more effective than red at retarding curvature; when seedlings were irradiated prior to geostimulus, only red light affected geocurvature. These results are interpreted as a further example of the kinetically distinct effects of red and blue light on hypocotyl development. Blue light elicited a rapid, immediate response effective only during the period of irradiation; red light induced a response characterized by a lag period and persistence in subsequent darkness. Etiolated mustard seedlings showed similar responses to light and gravity. The results are discussed in relation to the possibility that two photosystems operate in hypocotyl growth.     

Arabidopsis mutants resistant to S(+)-beta-methyl-alpha, beta-diaminopropionic acid, a cycad-derived glutamate receptor agonist

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that are the predominant neuroreceptors in the mammalian brain. Genes with high sequence similarity to animal iGluRs have been identified in Arabidopsis. To understand the role of Arabidopsis glutamate receptor-like (AtGLR) genes in plants, we have taken a pharmacological approach by examining the effects of BMAA [S(+)-beta-methyl-alpha, beta-diaminopropionic acid], a cycad-derived iGluR agonist, on Arabidopsis morphogenesis. When applied to Arabidopsis seedlings, BMAA caused a 2- to 3-fold increase in hypocotyl elongation and inhibited cotyledon opening during early seedling development. The effect of BMAA on hypocotyl elongation is light specific. Furthermore, BMAA effects on early morphogenesis of Arabidopsis can be reversed by the simultaneous application of glutamate, the native iGluR agonist in animals. To determine the targets of BMAA action in Arabidopsis, a genetic screen was devised to isolate Arabidopsis mutants with a BMAA insensitive morphology (bim). When grown in the light on BMAA, bim mutants exhibited short hypocotyls compared with wild type. bim mutants were grouped into three classes based on their morphology when grown in the dark in the absence of BMAA. Class-I bim mutants have a normal, etiolated morphology, similar to wild-type plants. Class-II bim mutants have shorter hypocotyls and closed cotyledons when grown in the dark. Class-III bim mutants have short hypocotyls and open cotyledons when grown in the dark, resembling the previously characterized constitutively photomorphogenic mutants (cop, det, fus, and shy). Further analysis of the bim mutants should help define whether plant-derived iGluR agonists target glutamate receptor signaling pathways in plants.