The family of CONSTANS-like genes in the moss Physcomitrella patens

The CONSTANS (CO) gene plays a central role in the regulation of flowering time in Arabidopsis, and is the founding member of a family of 17 CO homologues. CO and CO homologues have been found in flowering plants, but not in yeast and animals. To address the question of the origin of CO, this gene family was analysed in the moss Physcomitrella patens, a phylogenetically distant organism. In Arabidopsis and rice, three classes of CO homologues exist. The same three classes were found in Physcomitrella, suggesting that this gene family has ancient origins in the plant kingdom. In Arabidopsis, CO and 5 other genes belong to Group 1. Since only three Group 1 genes were identified in Physcomitrella, the family of CO homologues appears to be smaller in Physcomitrella than in Arabidopsis, in agreement with observations made with other gene families. Further analysis demonstrated that the Physcomitrella Group 1 genes are most similar to Arabidopsis Group 1 genes COL3/COL4/COL5, which are closely related to, but distinct from CO. An essential feature of CO function in Arabidopsis is a circadian controlled rhythm of transcript abundance. The three closely related Physcomitrella Group 1 genes have diurnal expression patterns that are distinct from the pattern of CO expression, and that are mainly caused by direct light induction. Distinct diurnal expression patterns are also observed for CO homologues that are not involved in control of flowering time. Consistently, the Physcomitrella CO homologues are unable to promote flowering upon expression in Arabidopsis. Together, the findings indicate that the CO branch of Group 1 genes does not exist in Physcomitrella. The role of CO in flowering time control was possibly derived from an ancestral function of Group 1 genes in light signal transduction. The function of the three Physcomitrella CO homologues was studied by exploiting the feasibility of gene targeting. A disruptant was generated for each Group 1 CO homologue in Physcomitrella, whereas in Arabidopsis only CO has been inactivated to date. Phenotypical analysis of the disruptants revealed no developmental defects, nor an alteration of the phototropic growth response. The high degree of sequence conservation between the three genes and the similar expression patterns suggest redundancy. Therefore, simultaneous inactivation of all three genes may be necessary to elucidate their function

Certification Report - The certification of different mass fractions of DAS-81419-2 in soya seed powder: Certified Reference Materials ERM®-BF437a, ERM®-BF437b, ERM®-BF437c, ERM®-BF437d and ERM®-BF437e

This report describes the production of a set of Certified Reference Materials (CRMs) ERM BF437a, b, c, d and e, which are certified for their DAS-81419-2 mass fractions. This material was produced following ISO Guide 34:2009 and is certified in accordance with ISO Guide 35:2006. Genetically modified (GM) seeds of the soya event DAS-81419-2 and seeds from a non-GM soya variety were milled to obtain GM and non-GM seed powders with a similar particle size distribution. Mixtures of non-GM and GM soya powder were prepared gravimetrically. The certified value was obtained from the gravimetric preparations, taking into account the genetic purity with respect to the event DAS-81419-2 of the two powder materials and their water mass fractions. The certified values were confirmed by event-specific real-time PCR as an independent verification method (measurements were within the scope of accreditation to ISO/IEC 17025:2005). Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties relating to possible inhomogeneity (Section 4), instability (Section 5) and characterisation (Section 6). The materials are intended for the calibration or quality control of real-time PCR measurements to identify DAS-81419-2 soya and/or quantify its mass fraction. As with any reference material, they can also be used for establishing control charts or for carrying out validation studies. The CRMs are available in glass vials containing at least 1 g of dried soya seed powder which were sealed under atmosphere of argon. The minimum amount of sample to be used is 200 mg. The CRMs were accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortiumJRC.D.2-Standards for Innovation and sustainable Developmen

CERTIFICATION REPORT The certification of different mass fractions of DP-ØØ4114-3 in maize seed powder Certified Reference Materials ERM®-BF439a, ERM®-BF439b, ERM®-BF439c, ERM®-BF439d and ERM®-BF439e

This report describes the production of a set of Certified Reference Materials (CRMs) ERM BF439a, b, c, d and e, which are certified for their 4114 maize event mass fractions. These materials were produced following ISO Guide 34:2009 and are certified in accordance with ISO Guide 35:2006. The materials are intended for the calibration or quality control of real-time PCR measurements to identify 4114 maize and/or quantify its mass fraction. As with any reference material, they can also be used for establishing control charts or for carrying out validation studies. The CRMs were accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Standards for Innovation and sustainable Developmen

SBP-domain transcription factors as possible effectors of cryptochrome-mediated blue light signalling in the moss Physcomitrella patens

Cryptochromes are blue light absorbing photoreceptors found in many organisms and involved in numerous developmental processes. At least two highly similar cryptochromes are known to affect branching during gametophytic development in the moss Physcomitrella patens. We uncovered a relationship between these cryptochromes and the expression of particular members of the SBP-box genes, a plant specific transcription factor family. Transcript levels of the respective moss SBP-box genes, all belonging to the LG1-subfamily, were found to be dependent, albeit not exclusively, on blue light. Moreover, disruptant lines generated for two moss representatives of this SBP-box gene subfamily, both showed enhanced caulonema side branch formation, a phenotype opposite to that of the ppcry1a/1b double disruptant line. In this report we show that PpCRY1a and PpCRY1b act negatively on the transcript levels of several related moss SBP-box genes and that at least PpSBP1 and PpSBP4 act as negative regulators of side branch formation

Boosting heterologous protein production in transgenic dicotyledonous seeds using Phaseolus vulgaris regulatory sequences

Over the past decade, several high-value proteins have been produced in different transgenic plant tissues such as leaves, tubers, and seeds(1,2). Despite recent advances, many heterologous proteins accumulate to low concentrations, and the optimization of expression cassettes to make in planta production and purification economically feasible remains critical. Here, the regulatory sequences of the seed storage protein gene arcelin 5-I (arc5-I) of common bean (Phaseolus vulgaris)(3) were evaluated for producing heterologous proteins in dicotyledonous seeds. The murine single-chain variable fragment (scFv) G4 (ref. 4) was chosen as model protein because of the current industrial interest in producing antibodies and derived fragments in crops(5,6). In transgenic Arabidopsis thaliana seed stocks, the scFv under control of the 35S promoter of the cauliflower mosaic virus (CaMV) accumulated to approximately 1% of total soluble protein (TSP). However, a set of seed storage promoter constructs boosted the scFv accumulation to exceptionally high concentrations, reaching no less than 36.5% of TSP in homozygous seeds. Even at these high concentrations, the scFv proteins had antigen-binding activity and affinity similar to those produced in Escherichia coli. The feasibility of heterologous protein production under control of arc5-I regulatory sequences was also demonstrated in Phaseolus acutifolius, a promising crop for large-scale production