Design in Arabidopsis thaliana of a synchronous system of floral induction by one long day

A system of one-shot induction of flowering in Arabidopsis thaliana, ecotype Columbia, is described. Plants from vernalized seeds are grown for 2 months in 8 h short days at an irradiance of 48 µmol m^?2 sec^?1 (fluorescent light only). At that age they can be induced to flower by exposure to either a single long day or a single displaced short day. Non-induced plants stay vegetative for at least a further month. Synchrony of induction among the individuals of the population exposed to one long day is of the same order as in the best classical model plants, that is, the fastest individuals are only 6 h ahead of the slowest ones. A further advantage of this system is the large size of plants at the time of induction, allowing easy analysis of changes in leaves, leaf exudate and shoot meristem. The design of such a synchronous system will allow the timings of gene activations and deactivations to be established in the different plant parts, before flowers are initiated.     

The role of carbohydrates in the induction of flowering in Arabidopsis thaliana: comparison between the wild type and a starchless mutant

In order to test whether an increased export of carbohydrates by leaves and starch mobilization are critical for floral transition in Arabidopsis thaliana, the Columbia ecotype as well as its starchless mutant pgm and starch-in-excess mutant sex1 were investigated. Induction of flowering was achieved by exposure of plants to either one long day (LD) or one displaced short day (DSD). The following conclusions were drawn: (i) Both the pgm and sex1 mutants have a late-flowering phenotype in days shorter than 16 h. (ii) When inductive treatments cause a large percentage of induced plants, there is always a large, early and transient increase in carbohydrate export from leaves. By contrast, when an inductive treatment results in only a low percentage of induced plants (pgm plants exposed to one DSD), the export of carbohydrates from leaves is not increased, supporting the idea that phloem carbohydrates have a critical function in floral transition. (iii) Starch mobilization is not required to obtain an increased carbohydrate export when induction is by one LD (extended period of photosynthesis), but is absolutely essential when induction is by one DSD (period of photosynthesis unaffected). (iv) Floral induction apparently increases the capability of the leaf phloem-loading system. Received: 27 August 1997 / Accepted: 6 March 1998     

The Rhizobium meliloti region located downstream of the nod box n6 is involved in the specific nodulation of Medicago lupulina

Abstract Six nod box regulatory sequences are present in the Rhizobium meliloti genome. We have analysed the DNA region located downstream of nod box n6, and identified three open reading frames, designated nolQa, nolQb and nolS . LacZ fusions in these ORFs are not induced by classical nod gene inducers, which indicates that their expression either is not under the control of the nod box, or involves another regulatory mechanism acting in conjunction with the NodD/nod box regulatory circuit. Mutations in this n6 locus result in a delay in nodule formation on a particular host, Medicago lupulina . As this region is not strictly conserved among different R. meliloti strains, nolQa, nolQb and nolS may constitute auxiliary nodulation genes, for which the selection pressure is limited to particular host plants.     

Cryo-EM reveals conformational flexibility in apo DNA polymerase ζ

The translesion synthesis (TLS) DNA polymerases Rev1 and Polζ function together in DNA lesion bypass during DNA replication, acting as nucleotide inserter and extender polymerases, respectively. While the structural characterization of the Saccharomyces cerevisiae Polζ in its DNA-bound state has illuminated how this enzyme synthesizes DNA, a mechanistic understanding of TLS also requires probing conformational changes associated with DNA- and Rev1 binding. Here, we used single-particle cryo-electron microscopy to determine the structure of the apo Polζ holoenzyme. We show that compared with its DNA-bound state, apo Polζ displays enhanced flexibility that correlates with concerted motions associated with expansion of the Polζ DNA-binding channel upon DNA binding. We also identified a lysine residue that obstructs the DNA-binding channel in apo Polζ, suggesting a gating mechanism. The Polζ subunit Rev7 is a hub protein that directly binds Rev1 and is a component of several other protein complexes such as the shieldin DNA double-strand break repair complex. We analyzed the molecular interactions of budding yeast Rev7 in the context of Polζ and those of human Rev7 in the context of shieldin using a crystal structure of Rev7 bound to a fragment of the shieldin-3 protein. Overall, our study provides new insights into Polζ mechanism of action and the manner in which Rev7 recognizes partner proteins.     

Detection of glycoalkaloids using disposable biosensors based on genetically modified enzymes

In this work we present a rapid, selective, and highly sensitive detection of α-solanine and α-chaconine using cholinesterase-based sensors. The high sensitivity of the devices is brought by the use of a genetically modified acetylcholinesterase (AChE), combined with a one-step detection method based on the measurement of inhibition slope. The selectivity was obtained by using butyrylcholinesterase (BChE), an enzyme able to detect these two toxins with differential inhibition kinetics. The enzymes were immobilized via entrapment in PVA-AWP polymer directly on the working electrode surface. The analysis of the resulting inhibition slope was performed employing linear regression function included in Matlab. The high toxicity of α-chaconine compared to α-solanine due to a better affinity to the active site was proved. The inhibition of glycoalkaloids (GAs) mixture was performed over AChE enzyme wild-type AChE and BChE biosensors resulting in the detection of synergism effect. The developed method allows the detection of (GAs) at 50 ppb in potato matrix.     

Discoidin domain receptor 1 controls linear invadosome formation via a Cdc42–Tuba pathway

Accumulation of type I collagen fibrils in tumors is associated with an increased risk of metastasis. Invadosomes are F-actin structures able to degrade the extracellular matrix. We previously found that collagen I fibrils induced the formation of peculiar linear invadosomes in an unexpected integrin-independent manner. Here, we show that Discoidin Domain Receptor 1 (DDR1), a collagen receptor overexpressed in cancer, colocalizes with linear invadosomes in tumor cells and is required for their formation and matrix degradation ability. Unexpectedly, DDR1 kinase activity is not required for invadosome formation or activity, nor is Src tyrosine kinase. We show that the RhoGTPase Cdc42 is activated on collagen in a DDR1-dependent manner. Cdc42 and its specific guanine nucleotide-exchange factor (GEF), Tuba, localize to linear invadosomes, and both are required for linear invadosome formation. Finally, DDR1 depletion blocked cell invasion in a collagen gel. Altogether, our data uncover an important role for DDR1, acting through Tuba and Cdc42, in proteolysis-based cell invasion in a collagen-rich environment.