Internal translation initiation in the mRNA for the Neurospora crassa albino-3 gene

The "ribosome scanning model" for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of multiple translational start sites on the mRNA of the Neurospora crassa gene albino-3 (al-3), encoding the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthase. This was revealed by the molecular analysis of an al-3 mutant carrying a deletion within the coding sequence, which was expected to prevent the synthesis of a functional geranylgeranyl-pyrophosphate synthase because of ribosome frameshifting and premature translational termination. However, the mutants could maintain appreciable geranylgeranyl-pyrophosphate synthase activity through a mechanism operating at the translational level, whereby a fraction of ribosomes initiated protein synthesis from either of two internal in-frame AUG codons located downstream of the deletion, thus producing a shortened but still active version of the geranylgeranyl-pyrophosphate synthase. The results presented indicate that the internal AUG codons are recognized mainly or solely by direct ribosome binding rather than by "leaky scanning" from the 5' end of the mRNA.The "ribosome scanning model" for translational initiation predicts that eukaryotic mRNAs should, as a rule, be monocistronic. However, cases have recently been described of eukaryotic mRNAs producing more than one protein through alternative translational initiation at several different AUG codons. The present work reports the occurrence of multiple translational start sites on the mRNA of the Neurospora crassa gene albino-3 (al-3), encoding the carotenoid biosynthetic enzyme geranylgeranyl-pyrophosphate synthase. This was revealed by the molecular analysis of an al-3 mutant carrying a deletion within the coding sequence, which was expected to prevent the synthesis of a functional geranylgeranyl-pyrophosphate synthase because of ribosome frameshifting and premature translational termination. However, the mutants could maintain appreciable geranylgeranyl-pyrophosphate synthase activity through a mechanism operating at the translational level, whereby a fraction of ribosomes initiated protein synthesis from either of two internal in-frame AUG codons located downstream of the deletion, thus producing a shortened but still active version of the geranylgeranyl-pyrophosphate synthase. The results presented indicate that the internal AUG codons are recognized mainly or solely by direct ribosome binding rather than by "leaky scanning" from the 5' end of the mRNA

“White Collar 1, a central regulator of blue light response in Neurospora, is a Zinc finger protein”

The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway. We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation.The Neurospora crassa blind mutant white collar-1 (wc-1) is pleiotropically defective in all blue light-induced phenomena, establishing a role for the wc-1 gene product in the signal transduction pathway. We report the cloning of the wc-1 gene isolated by chromosome walking and mutant complementation. The elucidation of the wc-1 gene product provides a key piece of the blue light signal transduction puzzle. The wc-1 gene encodes a 125 kDa protein whose encoded motifs include a single class four, zinc finger DNA binding domain and a glutamine-rich putative transcription activation domain. We demonstrate that the wc-1 zinc finger domain, expressed in Escherichia coli, is able to bind specifically to the promoter of a blue light-regulated gene of Neurospora using an in vitro gel retardation assay. Furthermore, we show that wc-1 gene expression is autoregulated and is transcriptionally induced by blue light irradiation

DOF AFFECTING GERMINATION 2 is a positive regulator of light-mediated seed germination and is repressed by DOF AFFECTING GERMINATION 1

Abstract BACKGROUND: The transcription factor DOF AFFECTING GERMINATION1 (DAG1) is a repressor of the light-mediated seed germination process. DAG1 acts downstream PHYTOCHROME INTERACTING FACTOR3-LIKE 5 (PIL5), the master repressor, and negatively regulates gibberellin biosynthesis by directly repressing the biosynthetic gene AtGA3ox1. The Dof protein DOF AFFECTING GERMINATION (DAG2) shares a high degree of aminoacidic identity with DAG1. While DAG1 inactivation considerably increases the germination capability of seeds, the dag2 mutant has seeds with a germination potential substantially lower than the wild-type, indicating that these factors may play opposite roles in seed germination. RESULTS: We show here that DAG2 expression is positively regulated by environmental factors triggering germination, whereas its expression is repressed by PIL5 and DAG1; by Chromatin Immuno Precipitation (ChIP) analysis we prove that DAG1 directly regulates DAG2. In addition, we show that Red light significantly reduces germination of dag2 mutant seeds. CONCLUSIONS: In agreement with the seed germination phenotype of the dag2 mutant previously published, the present data prove that DAG2 is a positive regulator of the light-mediated seed germination process, and particularly reveal that this protein plays its main role downstream of PIL5 and DAG1 in the phytochrome B (phyB)-mediated pathway

The DAG1 transcription factor negatively regulates the seed-to-seedling transition in Arabidopsis acting on ABA and GA levels

BACKGROUND: In seeds, the transition from dormancy to germination is regulated by abscisic acid (ABA) and gibberellins (GAs), and involves chromatin remodelling. Particularly, the repressive mark H3K27 trimethylation (H3K27me3) has been shown to target many master regulators of this transition. DAG1 (DOF AFFECTING GERMINATION1), is a negative regulator of seed germination in Arabidopsis, and directly represses the GA biosynthetic gene GA3ox1 (gibberellin 3-β-dioxygenase 1). We set to investigate the role of DAG1 in seed dormancy and maturation with respect to epigenetic and hormonal control. RESULTS: We show that DAG1 expression is controlled at the epigenetic level through the H3K27me3 mark during the seed-to-seedling transition, and that DAG1 directly represses also the ABA catabolic gene CYP707A2; consistently, the ABA level is lower while the GA level is higher in dag1 mutant seeds. Furthermore, both DAG1 expression and protein stability are controlled by GAs. CONCLUSIONS: Our results point to DAG1 as a key player in the control of the developmental switch between seed dormancy and germination

The COP9 SIGNALOSOME is required for postembryonic meristem maintenance in Arabidopsis thaliana

Cullin-RING E3 ligases (CRLs) regulate different aspects of plant development, and are activated by modification of their cullin subunit with the ubiquitin-like protein NEDD8 (NEural precursor cell expressed Developmentally Down-regulated 8) (neddylation) and deactivated by NEDD8 removal (deneddylation). The CONSTITUTIVELY PHOTOMORPHOGENIC9 (COP9) signalosome (CSN) acts as a molecular switch of CRLs activity by reverting their neddylation status, but its contribution to embryonic and early seedling development remains poorly characterized. Here, we analyzed the phenotypic defects of csn mutants and monitored the cullin deneddylation/neddylation ratio during embryonic and early seedling development. We show that while csn mutants can complete embryogenesis (albeit at a slower pace than wild type) and are able to germinate (albeit at a reduced rate), they progressively loose meristem activity upon germination, until they become unable to sustain growth. We also show that the majority of cullin proteins is progressively neddylated during the late stages of seed maturation and becomes deneddylated upon seed germination. This developmentally regulated shift in the cullin neddylation status is absent in csn mutants. We conclude that the CSN and its cullin deneddylation activity are required to sustain postembryonic meristem function in Arabidopsis

The DOF Transcription Factors in Seed and Seedling Development

The DOF (DNA binding with one finger) family of plant-specific transcription factors (TF) was first identified in maize in 1995. Since then, DOF proteins have been shown to be present in the whole plant kingdom, including the unicellular alga Chlamydomonas reinhardtii. The DOF TF family is characterised by a highly conserved DNA binding domain (DOF domain), consisting of a CX2C-X21-CX2C motif, which is able to form a zinc finger structure. Early in the study of DOF proteins, their relevance for seed biology became clear. Indeed, the PROLAMIN BINDING FACTOR (PBF), one of the first DOF proteins characterised, controls the endosperm-specific expression of the zein genes in maize. Subsequently, several DOF proteins from both monocots and dicots have been shown to be primarily involved in seed development, dormancy and germination, as well as in seedling development and other light-mediated processes. In the last two decades, the molecular network underlying these processes have been outlined, and the main molecular players and their interactions have been identified. In this review, we will focus on the DOF TFs involved in these molecular networks, and on their interaction with other proteins

Inhibition of Polycomb Repressive Complex2 activity reduces trimethylation of H3K27 and affects development in Arabidopsis seedlings

Background: Polycomb repressive complex 2 (PRC2) is an epigenetic transcriptional repression system, whose catalytic subunit (ENHANCER OF ZESTE HOMOLOG 2, EZH2 in animals) is responsible for trimethylating histone H3 at lysine 27 (H3K27me3). In mammals, gain-of-function mutations as well as overexpression of EZH2 have been associated with several tumors, therefore making this subunit a suitable target for the development of selective inhibitors. Indeed, highly specific small-molecule inhibitors of EZH2 have been reported. In plants, mutations in some PRC2 components lead to embryonic lethality, but no trial with any inhibitor has ever been reported. Results: We show here that the 1,5-bis (3-bromo-4-methoxyphenyl)penta-1,4-dien-3-one compound (RDS 3434), previously reported as an EZH2 inhibitor in human leukemia cells, is active on the Arabidopsis catalytic subunit of PRC2, since treatment with the drug reduces the total amount of H3K27me3 in a dose-dependent fashion. Consistently, we show that the expression level of two PRC2 targets is significantly increased following treatment with the RDS 3434 compound. Finally, we show that impairment of H3K27 trimethylation in Arabidopsis seeds and seedlings affects both seed germination and root growth. Conclusions: Our results provide a useful tool for the plant community in investigating how PRC2 affects transcriptional control in plant development

A PHABULOSA-Controlled Genetic Pathway Regulates Ground Tissue Patterning in the Arabidopsis Root

In both animals and plants, development involves anatomical modifications. In the root of Arabidopsis thaliana, maturation of the ground tissue (GT)—a tissue comprising all cells between epidermal and vascular ones—is a paradigmatic example of these modifications, as it generates an additional tissue layer, the middle cortex (MC).1, 2, 3, 4 In early post-embryonic phases, the Arabidopsis root GT is composed of one layer of endodermis and one of cortex. A second cortex layer, the MC, is generated by asymmetric cell divisions in about 80% of Arabidopsis primary roots, in a time window spanning from 7 to 14 days post-germination (dpg). The cell cycle regulator CYCLIN D6;1 (CYCD6;1) plays a central role in this process, as its accumulation in the endodermis triggers the formation of MC.5 The phytohormone gibberellin (GA) is a key regulator of the timing of MC formation, as alterations in its signaling and homeostasis result in precocious endodermal asymmetric cell divisions.3,6,7 However, little is known on how GAs are regulated during GT maturation. Here, we show that the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) transcription factor PHABULOSA (PHB) is a master regulator of MC formation, controlling the accumulation of CYCD6;1 in the endodermis in a cell non-autonomous manner. We show that PHB activates the GA catabolic gene GIBBERELLIN 2 OXIDASE 2 (GA2ox2) in the vascular tissue, thus regulating the stability of the DELLA protein GIBBERELLIN INSENSITIVE (GAI)—a GA signaling repressor—in the root and, hence, CYCD6;1 expression in the endodermis

La germinazione dei semi

Seed germination is a plant developmental process defined by the protrusion of the radicle from the seed coat. The seed is a quiescent organ, which represents an evolutionary successful method of sexual reproduction in vascular plants. The earliest seed plants, "progymnosperms", emerged in the late Devonian about 370 million years ago. Once embryogenesis is completed, the mature seed is dehydrated and subsequently becomes dormant. Seed dormancy could be considered simply as a block to the completion of germination of an intact viable seed under favourable conditions. Therefore seed germination begins with rehydration (imbibition) of seeds and is controlled by multiple environmental and endogenous factors like temperature, light, and the phytohormones gibberellins (GA) and abscissic acid (ABA). GA and ABA respectively promotes and inhibits this process. Germination of seeds of most annuals needs a pulse of R light and is mediated mainly by the photoreceptor phyB. Recently it has been partially uncovered the transduction pathway leading to seed germination in Arabidopsis thaliana. PIL5 (Phytochrome Interacting Factor3- Like 5) plays a pivotal role by negatively regulating this process. PIL5 activates transcription of the target genes GA- INSENSITIVE (GAI) and REPRESSOR OF GA1-3 (RGA), whereas indirectly regulates GA and ABA metabolism. The DAG1 Dof (DNA binding with One Finger) protein has been previously characterized as a transcription factor primarily involved in the negative control of seed germination. Recently it has been demonstrated that DAG1 is part of the phyB/PIL5 signal transduction pathway leading to seed germination. In fact, DAG1 negatively regulates GA biosynthesis by directly binding the AtGA3ox1 promote