Degradation of MONOCULM 1 by APC/CTAD1 regulates rice tillering

A rice tiller is a specialized grain-bearing branch that contributes greatly to grain yield. The MONOCULM 1 (MOC1) gene is the first identified key regulator controlling rice tiller number; however, the underlying mechanism remains to be elucidated. Here we report a novel rice gene, Tillering and Dwarf 1 (TAD1), which encodes a co-activator of the anaphase-promoting complex (APC/C), a multi-subunit E3 ligase. Although the elucidation of co-activators and individual subunits of plant APC/C involved in regulating plant development have emerged recently, the understanding of whether and how this large cell-cycle machinery controls plant development is still very limited. Our study demonstrates that TAD1 interacts with MOC1, forms a complex with OsAPC10 and functions as a co-activator of APC/C to target MOC1 for degradation in a cell-cycle-dependent manner. Our findings uncovered a new mechanism underlying shoot branching and shed light on the understanding of how the cell-cycle machinery regulates plant architecture

Rice APC/CTE controls tillering by mediating the degradation of MONOCULM 1

Rice MONOCULM 1 (MOC1) and its orthologues LS/LAS (lateral suppressor in tomato and Arabidopsis) are key promoting factors of shoot branching and tillering in higher plants. However, the molecular mechanisms regulating MOC1/LS/LAS have remained elusive. Here we show that the rice tiller enhancer (te) mutant displays a drastically increased tiller number. We demonstrate that TE encodes a rice homologue of Cdh1, and that TE acts as an activator of the anaphase promoting complex/cyclosome (APC/C) complex. We show that TE coexpresses with MOC1 in the axil of leaves, where the APC/CTE complex mediates the degradation of MOC1 by the ubiquitin–26S proteasome pathway, and consequently downregulates the expression of the meristem identity gene Oryza sativa homeobox 1, thus repressing axillary meristem initiation and formation. We conclude that besides having a conserved role in regulating cell cycle, APC/CTE has a unique function in regulating the plant-specific postembryonic shoot branching and tillering, which are major determinants of plant architecture and grain yield

Regulation of the Fruit-Specific PEP Carboxylase SlPPC2 Promoter at Early Stages of Tomato Fruit Development

The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the β-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5′ SlPPC2 promoter deletions fused to LUC in fruits at the 8th day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5′ UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars

The anaphase promoting complex activator CCS52A, a key factor for fruit growth and endoreduplication in tomato

Tomato fruit growth is characterized by the occurrence of numerous rounds of DNA endoreduplication in connection to cell expansion and final fruit size determination. Endoreduplication occurs as an impairment of mitosis, which can originate from the selective degradation of M-phase-specific cyclins via the ubiquitin-mediated proteolytic pathway, requiring the E3 ubiquitin ligase Anaphase Promoting Complex/Cyclosome (APC/C). In plants CCS52A is the ortholog of CDH1/FZR proteins from yeast, drosophila and human, belonging to the WD40-repeat protein family. During fruit development, the SlCCS52A gene expression is specifically associated to endoreduplication in tomato. Altering SlCCS52A expression in either negative or positive manner impacts the extent of endoreduplication in fruit and affects fruit size. When SlCCS52A is down-expressed endoreduplication is impaired during fruit growth leading to reduced fruit growth. However when SlCCS52A is overexpressed, endoreduplication is initially delayed, accounting for the altered final fruit size, but resumes and is even enhanced leading to fruit growth recovery, pointing at the physiological role of endoreduplication in growth induction during tomato fruit development

Functional analysis of the anaphase promoting complex activator CCS52A highlights the crucial role of endo-reduplication for fruit growth in tomato

International audienceTomato fruit growth is characterized by the occurrence of numerous rounds of DNA endo‐reduplication in connection with cell expansion and final fruit size determination. Endo‐reduplication is an impairment of mitosis that originates from the selective degradation of M phase‐specific cyclins via the ubiquitin‐mediated proteolytic pathway, requiring the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C). Two types of APC/C activators, namely CCS52 and CDC20 proteins, exist in plants. We report here the molecular characterization of such APC/C activators during fruit development, and provide an in planta functional analysis of SlCCS52A, a gene that is specifically associated with endo‐reduplication in tomato. Altering SlCCS52A expression in either a negative or positive manner had an impact on the extent of endo‐reduplication in fruit, and fruit size was reduced in both cases. In SlCCS52A over‐expressing fruits, endo‐reduplication was initially delayed, accounting for the altered final fruit size, but resumed and was even enhanced at 15 days post anthesis (dpa), leading to fruit growth recovery. This induction of growth mediated by endo‐reduplication had a considerable impact on nitrogen metabolism in developing fruits. Our data contribute to unravelling of the physiological role of endo‐reduplication in growth induction during tomato fruit development

Biochemical Characterization of Human Anti- Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum

International audienceMonoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharma-ceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C-and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro-and microhetero-geneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans