Arabidopsis COP8, COP10, and COP11 genes are involved in repression of photomorphogenic development in darkness.

Wild-type Arabidopsis seedlings are capable of following two developmental programs: photomorphogenesis in the light and skotomorphogenesis in darkness. Screening of Arabidopsis mutants for constitutive photomorphogenic development in darkness resulted in the identification of three new loci designated COP8, COP10, and COP11. Detailed examination of the temporal morphological and cellular differentiation patterns of wild-type and mutant seedlings revealed that in darkness, seedlings homozygous for recessive mutations in COP8, COP10, and COP11 failed to suppress the photomorphogenic developmental pathway and were unable to initiate skotomorphogenesis. As a consequence, the mutant seedlings grown in the dark had short hypocotyls and open and expanded cotyledons, with characteristic photomorphogenic cellular differentiation patterns and elevated levels of light-inducible gene expression. In addition, plastids of dark-grown mutants were defective in etioplast differentiation. Similar to cop1 and cop9, and in contrast to det1 (deetiolated), these new mutants lacked dark-adaptive change of light-regulated gene expression and retained normal phytochrome control of seed germination. Epistatic analyses with the long hypocotyl hy1, hy2, hy3, hy4, and hy5 mutations suggested that these three loci, similar to COP1 and COP9, act downstream of both phytochromes and a blue light receptor, and probably HY5 as well. Further, cop8-1, cop10-1, and cop11-1 mutants accumulated higher levels of COP1, a feature similar to the cop9-1 mutant. These results suggested that COP8, COP10, and COP11, together with COP1, COP9, and DET1, function to suppress the photomorphogenic developmental program and to promote skotomorphogenesis in darkness. The identical phenotypes resulting from mutations in COP8, COP9, COP10, and COP11 imply that their encoded products function in close proximity, possibly with some of them as a complex, in the same signal transduction pathway.     

Endogenous Abscisic Acid Promotes Hypocotyl Growth and Affects Endoreduplication during Dark-Induced Growth in Tomato (Solanum lycopersicum L.)

Dark-induced growth (skotomorphogenesis) is primarily characterized by rapid elongation of the hypocotyl. We have studied the role of abscisic acid (ABA) during the development of young tomato (Solanum lycopersicum L.) seedlings. We observed that ABA deficiency caused a reduction in hypocotyl growth at the level of cell elongation and that the growth in ABA-deficient plants could be improved by treatment with exogenous ABA, through which the plants show a concentration dependent response. In addition, ABA accumulated in dark-grown tomato seedlings that grew rapidly, whereas seedlings grown under blue light exhibited low growth rates and accumulated less ABA. We demonstrated that ABA promotes DNA endoreduplication by enhancing the expression of the genes encoding inhibitors of cyclin-dependent kinases SlKRP1 and SlKRP3 and by reducing cytokinin levels. These data were supported by the expression analysis of the genes which encode enzymes involved in ABA and CK metabolism. Our results show that ABA is essential for the process of hypocotyl elongation and that appropriate control of the endogenous level of ABA is required in order to drive the growth of etiolated seedlings.     

Genetic and developmental control of nuclear accumulation of COP1, a repressor of photomorphogenesis in Arabidopsis.

Using a beta-glucuronidase (GUS) reporter-COP1 fusion transgene, it was shown previously that Arabidopsis COP1 acts within the nucleus as a repressor of seedling photomorphogenic development and that high inactivation of COP1 was accompanied by a reduction of COP1 nuclear abundance (A.G. von Arnim, X.-W. Deng [1994] Cell 79: 1035-1045). Here we report that the GUS-COP1 fusion transgene can completely rescue the defect of cop1 mutations and thus is fully functional during seedling development. The kinetics of GUS-COP1 relocalization in a cop1 null mutant background during dark/light transitions imply that the regulation of the functional nuclear COP1 level plays a role in stably maintaining a committed seedling's developmental fate rather than in causing such a commitment. Analysis of GUS-COP1 cellular localization in mutant hypocotyls of all pleiotropic COP/DET/FUS loci revealed that nuclear localization of GUS-COP1 was diminished under both dark and light conditions in all mutants tested, whereas nuclear localization was not affected in the less pleiotropic cop4 mutant. Using both the brassinosteroid-deficient mutant det2 and brassinosteroid treatment of wild-type seedlings, we have demonstrated that brassinosteroid does not control the hypocotyl cell elongation through regulation nuclear localization of COP1. The growth regulator cytokinin, which also dramatically reduced hypocotyl cell elongation in the absence of light, did not prevent GUS-COP1 nuclear localization in dark-grown seedlings. Our results suggest that all of the previously characterized pleiotropic COP/DET/FUS loci are required for the proper nuclear localization of the COP1 protein in the dark, whereas the less pleiotropic COP/DET loci or plant regulators tested are likely to act either downstream of COP1 or by independent pathways.     

HFR1, a phytochrome A-signalling component,acts in a separate pathway from HY5, downstream of COP1 in Arabidopsis thaliana

HFR1, a basic helix-loop-helix protein, is known to be required for a subset of phytochrome A (phyA)-dependent photoresponses. To investigate the role of HFR1 in light signalling, we have examined the genetic interaction between HFR1 and HY5, a positive regulator of light signalling, and COP1, a repressor of photomorphogenesis. Double mutant analysis suggests that HFR1 mediates phyA-dependent inhibition of hypocotyl elongation independently of HY5. HFR1 was shown to be necessary for a subset of cop1-triggered photomorphogenic phenotypes in the dark, including inhibition of hypocotyl elongation, gravitropic hypocotyl growth, and expression of the light-inducible genes CAB and RBCS. Phenotypic analysis of the triple mutant cop1hy5hfr1 indicated that both HFR1 and HY5 are required for cop1-mediated photomorphogenic seedling development in darkness, consistent with their additive roles in phyA-dependent signalling. Taken together, these results suggest that HFR1 might act downstream of COP1, in a separate pathway from HY5, to mediate photomorphogenesis in Arabidopsis.     

Genetic interactions between the chlorate-resistant mutant cr 8 8 and the photomorphogenic mutants cop1 and hy5

The chlorate-resistant mutant cr88 is defective in photomorphogenesis, as shown by the phenotypes of long hypocotyls in red light and yellow cotyledons under all light conditions. A subset of light-regulated genes is expressed at subnormal levels in cr88. To analyze further the role that CR88 plays in photomorphogenesis, we investigated the genetic interactions between cr88 and mutants of two other loci affecting photomorphogenesis, CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and LONG HYPOCOTYL5 (HY5). COP1 represses the expression of light-regulated genes in the dark, and HY5 inhibits hypocotyl elongation in the light. Using morphological, cellular, and gene expression criteria for epistasis analyses to position CR88 in the genetic hierarchy of the photomorphogenesis pathway, we determined that CR88 acts downstream of COP1 but in a branch separate from HY5. In the course of our analysis, we discovered that light causes extensive destruction of plastids in dark-grown cop1 seedlings and that cr88 prevents this destruction.     

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.     

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.     

The effects of DELLAs on growth change with developmental stage and brassinosteroid levels

There are two stages in photomorphogenesis. First, seedlings detect light and open their cotyledons. Second, seedlings optimize their light environment by controlled elongation of the seedling stem or hypocotyl. In this study, we used time‐lapse imaging to investigate the relationship between the brassinosteroid (BR) and gibberellin (GA) hormones across both stages of photomorphogenesis. During the transition between one stage and the other, growth promotion by BRs and GAs switched from an additive to a synergistic relationship. Molecular genetic analysis revealed unexpected roles for known participants in the GA pathway during this period. Members of the DELLA family could either repress or enhance BR growth responses, depending on developmental stage. At the transition point for seedling growth dynamics, the BR and GA pathways had opposite effects on DELLA protein levels. In contrast to GA‐induced DELLA degradation, BR treatments increased the levels of REPRESSOR of ga1‐3 (RGA) and mimicked the molecular effects of stabilizing DELLAs. In addition, DELLAs showed complex regulation of genes involved in BR biosynthesis, implicating them in BR homeostasis. Growth promotion by GA alone depended on the PHYTOCHROME INTERACTING FACTOR (PIF) family of master growth regulators. The effects of BR, including the synergistic effects with GA, were largely independent of PIFs. These results point to a multi‐level, dynamic relationship between the BR and GA pathways.     

A New Class of Arabidopsis Constitutive Photomorphogenic Genes Involved in Regulating Cotyledon Development

Light signals have profound effects on morphogenesis of hypocotyls and cotyledons of Arabidopsis seedlings, but the mechanisms by which light signals are transduced and integrated to control these processes are poorly understood. We report here the identification of a new class of constitutive photomorphogenic (cop) mutants, cop2, cop3, and cop4, in which dark-grown seedlings have open and enlarged cotyledons resembling those of light-grown wild-type seedlings. The epistatic relationships of these three mutations to previously characterized phytochrome-deficient mutations suggest that COP2, COP3, and COP4 may act downstream of phytochrome in the light regulatory pathway. Mutations in each of the three loci alleviate the normal inhibition of cell-type differentiation, cell enlargement, and lateral cell division observed in cotyledons of dark-grown wild-type seedlings, but do not affect plastid differentiation. The cop4 mutation also leads to high-level dark expression of nuclear, but not plastid-encoded, light-inducible genes. We further show that for the nuclear cab1 gene encoding a chlorophyll a/b binding protein of the photosynthetic light-harvesting complex, activation in dark-grown cop4 mutants is achieved by modulation of promoter activity. Interestingly, COP4 modulates cab1 promoter activity through a pathway distinct from that of COP1 and COP9. Furthermore, cop4 mutants are defective in both root and shoot gravitropic responses, indicating that the COP4 locus may be involved in both light-signaling and gravity-sensing processes.     

Short communication: the N-terminal fragment of Arabidopsis photomorphogenic repressor COP1 maintains partial function and acts in a concentration-dependent manner

Arabidopsis seedlings exhibit distinct developmental patterns according to their light environment: photomorphogenesis in the light and etiolation or skotomorphogenesis in darkness. COP1 acts within the nucleus to repress photomorphogenesis in darkness, while light depletes COP1 from nucleus and abrogates this repression. COP1 contains three structural modules: a RING finger followed by a coiled-coil domain, and a WD40 repeat domain at the C-terminus. By introducing various domain deletion mutants of COP1 into cop1 null mutant backgrounds, we show that all three domains are essential for the function of COP1 in vivo. Interestingly, a fragment containing the N-terminal 282 amino acids of COP1 (N282) with both the RING finger and coiled-coil modules is sufficient to rescue the lethality of the cop1 null mutations at low expression level. However, high expression levels of the N282 fragment result in a phenocopy of the cop1 null mutation. The sensitivity of the seedling to levels of N282 could reflect the importance of the abundance of COP1 for the appropriate regulation of photomorphogenic development.     

COP1 re‐accumulates in the nucleus under shade

Shade‐avoider plants typically respond to shade‐light signals by increasing the rate of stem growth. CONSTITTUTIVE PHOTOMORPHOGENESIS 1 (COP1) is an E3 ligase involved in the ubiquitin labelling of proteins targeted for degradation. In dark‐grown seedlings, COP1 accumulates in the nucleus and light exposure causes COP1 migration to the cytosol. Here, we show that in Arabidopsis thaliana, COP1 accumulates in the nucleus under natural or simulated shade, despite the presence of far‐red light. In plants grown under white light, the transfer to shade‐light conditions triggers an unexpectedly rapid re‐accumulation of COP1 in the nucleus. The partial simulation of shade by lowering either blue or red light levels (maintaining far‐red light) caused COP1 nuclear re‐accumulation. Hypocotyl growth of wild‐type seedlings is more sensitive to afternoon shade than to morning shade. A residual response to shade was observed in the cop1 mutant background, but these seedlings showed inverted sensitivity as they responded to morning shade and not to afternoon shade. COP1 overexpression exaggerated the wild‐type pattern by enhancing afternoon sensitivity and making morning shade inhibitory of growth. COP1 nuclear re‐accumulation also responded more strongly to afternoon shade than to morning shade. These results are consistent with a signalling role of COP1 in shade avoidance. We propose a function of COP1 in setting the daily patterns of sensitivity to shade in the fluctuating light environments of plant canopies.     

Effect of light on ethylene production and hypocotyl growth of soybean seedlings

The apical 1-cm hypocotyl of dark-grown `Clark' soybean (Glycine max [L.] Merr.) seedlings produced ethylene at rates of 7 to 11 nanoliters per hour per gram when attached to the cotyledons. Such physiologically active rates occurred prior to the deceleration of hypocotyl elongation caused by the temperature of 25 C.

Daily exposure of the etiolated seedlings to red light promoted hypocotyl elongation and prevented its lateral swelling. Red light treatment also caused a 45% decrease in ethylene production. Far red irradiation following the red treatment reversed the red effects, suggesting that the ethylene intervenes as a regulator in the phytochrome control of `Clark' soybean hypocotyl growth at 25 C.

     

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.