Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga \u3ci\u3eNannochloropsis oceanica\u3c/i\u3e CCMP1779

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogendepleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus

Identification et caractérisation de deux nouveaux membres de la famille des inhibiteurs de pectine méthylesterase/invertase chez la tomate (Solanum lycopersion)

Les pectine méthylesterases et les invertases sont des enzymes clefs du métabolisme des glucides chez les plantes dont l'activité est modulée par des inhibiteurs appartenant à la même famille (PF04043) Dans ce travail, deux protéines de tomate appartenant à cette famille ont été identifiées et caractérisées Par RT-PCR quantitative, il a été montré que SolyPMEl est principalement exprimé dans le fruit rouge. Après expression en système hétérologue, purification et caractérisation, il s'agit du PME binding protein sans aucune activité mhibitnce. La purification de la protéine naturelle, par immuno-affinité, montre la formation d'un complexe avec la PME de tomate confirmant nos résultats précédents. SolyCIF a été exprimé dans Pichia postons et caractérisée d'un point de vue biochimique. SolyCIF est un inhibiteur d'invertase localise dans la paroi cellulaire et qui interagit in vitro avec l'invertase vacuolaire de tomate (TIV-1).AIX-MARSEILLE3-BU Sc.St Jérô (130552102) / SudocSudocFranceF

PECTIN METYLESTERASE INHIBITOR IN TOMATO FRUITS: CLONING AND MOLECULAR CHARACTERIZATION

Pectin is one of the main components of primary plant cell wall and its structure is critical for plant growth, intercellular adhesion, fruit texture and interaction with pathogens. Pectin is secreted in a highly methyl-esterified form and subsequently de-esterified by pectin methylesterases (PMEs) localized in the wall. PMEs take part in important physiological processes such as pollen growth and fruit ripening and are involved in the resistance response to fungal and viral pathogens. A mechanism of regulation of PME activity is played by specific proteinaceous inhibitors (PMEI). The structural bases of the interaction between PME from tomato and PMEI from kiwi fruit have been defined by solving the 3D structure of the PME/PMEI complex. We have identified and cloned a fruit-expressed cDNA of tomato with a significant sequence identity with kiwi PMEI. Structural alignment and modelling indicate that the encoded protein has the same fold of kiwi PMEI. Expression and functional characterization of the putative tomato PMEI will be presented

A functional pectin methylesterase inhibitor protein (SolyPMEI) is expressed during tomato fruit ripening and interacts with PME-1

A pectin methylesterase inhibitor (SolyPMEI) from tomato has been identified and characterised by a functional genomics approach. SolyPMEI is a cell wall protein sharing high similarity with Actinidia deliciosa PMEI (AdPMEI), the best characterised inhibitor from kiwi. It typically affects the activity of plant pectin methylesterases (PMEs) and is inactive against a microbial PME. SolyPMEI transcripts were mainly expressed in flower, pollen and ripe fruit where the protein accumulated at breaker and turning stages of ripening. The expression of SolyPMEI correlated during ripening with that of PME-1, the major fruit specific PME isoform. The interaction ofSolyPMEI with PME-1 was demonstrated in ripe fruit by gel filtration and by immunoaffinity chromatography. The analysis of the zonal distribution of PME activity and the co-localization of SolyPMEI with high esterified pectins suggest that SolyPMEI regulates the spatial patterning of distribution of esterified pectins in fruit

Molecular cloning, expression and characterization of a novel apoplastic invertase inhibitor from tomato (Solanum lycopersicum) and its use to purify a vacuolar invertase

Protein inhibitors are molecules secreted by many plants. In a functional genomics approach, an invertase inhibitor (SolyCIF) of Solanum lycopersicum was identified at the Solanaceae Cornell University data bank (www.sgn.cornell.edu). It was established that this inhibitor is expressed mainly in the leaves, flowers and green fruit of the plant and localized in the cell wall compartment. The SolyCIF cDNA was cloned by performing RTePCR, fully sequenced and heterologously expressed in Pichia pastoris X-33. The purified recombinant protein obtained by performing ion-exchange chromatography and gel filtration was further biochemically characterized and used to perform affinity chromatography. The latter step made it possible to purify natural vacuolar invertase (TIV-1), which showed high rates of catalytic activity (438.3 U mg1) and efficiently degraded saccharose (Km ¼ 6.4 mM, Vmax ¼ 2.9 mmol saccharose min1 and kcat ¼ 7.25 103 s1 at pH 4.9 and 37 C). The invertase activity was strongly inhibited in a dose-dependent manner by SolyCIF produced in P. pastoris. In addition, Gel-SDSePAGE analysis strongly suggests that TIV-1 was proteolyzed in planta and it was established that the fragments produced have to be tightly associated for its enzymatic activity to occur. We further investigated the location of the proteolytic sites by performing NH2-terminal Edman degradation on the fragments. The molecular model for TIV-1 shows that the fragmentation splits the catalytic site of the enzyme into two halves, which confirms that the enzymatic activity is possible only when the fragments are tightly associated

Molecular characterization of a novel apoplastic invertase inhibitor from tomato

Protein inhibitors are molecules secreted by many plants. In a functional genomics approach, an invertase inhibitor (SolyCIF) of Solanum lycopersicum was identified at the Solanaceae Cornell University data bank (www.sgn.cornell.edu). It was established that this inhibitor is expressed mainly in the leaves, flowers and green fruit of the plant and localized in the cell wall compartment. The SolyCIF cDNA was cloned by performing RT-PCR, fully sequenced and heterologously expressed in Pichia pastoris X-33. The purified recombinant protein obtained by performing ion-exchange chromatography and gel filtration was further biochemically characterized and used to perform affinity chromatography. The latter step made it possible to purify natural vacuolar invertase (TIV-1), which showed high rates of catalytic activity (438.3 U mg(-1)) and efficiently degraded saccharose (K-m = 6.4 mM, V-max = 2.9 mu mol saccharose min(-1) and k(cat) = 7.25 x 10(3) s(-1) at pH 4.9 and 37 degrees C). The invertase activity was strongly inhibited in a dose-dependent manner by SolyCIF produced in P. pastoris. In addition, Gel-SDS-PAGE analysis strongly suggests that TIV-1 was proteolyzed in planta and it was established that the fragments produced have to be tightly associated for its enzymatic activity to occur. We further investigated the location of the proteolytic sites by performing NH2-tenninal Edman degradation on the fragments. The molecular model for TIV-1 shows that the fragmentation splits the catalytic site of the enzyme into two halves, which confirms that the enzymatic activity is possible only when the fragments are tightly associated. (c) 2008 Elsevier Masson SAS. All rights reserved

Controlled expression of pectic enzymes in Arabidopsis thaliana enhances biomass conversion without adverse effects on growth

Lignocellulosic biomass from agriculture wastes is a potential source of biofuel, but its use is currently limited by the recalcitrance of the plant cell wall to enzymatic digestion. Modification of the wall structural components can be a viable strategy to overcome this bottleneck. We have previously shown that the expression of a fungal polygalacturonase (pga2 from Aspergillus niger) in Arabidopsis and tobacco plants reduces the levels of de-esterified homogalacturonan in the cell wall and significantly increases saccharification efficiency. However, plants expressing pga2 show stunted growth and reduced biomass production, likely as a consequence of an extensive loss of pectin integrity during the whole plant life cycle. We report here that the expression in Arabidopsis of another pectic enzyme, the pectate lyase 1 (PL1) of Pectobacterium carotovorum, under the control of a chemically inducible promoter, results, after induction of the transgene, in a saccharification efficiency similar to that of plants expressing pga2. However, lines with high levels of transgene induction show reduced growth even in the absence of the inducer. To overcome the problem of plant fitness, we have generated Arabidopsis plants that express pga2 under the control of the promoter of SAG12, a gene expressed only during senescence. These plants expressed pga2 only at late stages of development, and their growth was comparable to that of WT plants. Notably, leaves and stems of transgenic plants were more easily digested by cellulase, compared to WT plants, only during senescence. Expression of cell wall-degrading enzymes at the end of the plant life cycle may be therefore a useful strategy to engineer crops unimpaired in biomass yield but improved for bioconversion

O-Acetylation of Arabidopsis Hemicellulose Xyloglucan Requires AXY4 or AXY4L, Proteins with a TBL and DUF231 Domain[W][OA]

Most plant cell wall polysaccharides are O-acetylated. However, the acetyltransferases were elusive. Using a forward genetic approach, a putative xyloglucan O-acetyltransferase has now been identified in an unexpected gene family. This opens up future research into the identification of other O-acetyltransferases and the elucidation of the molecular mechanism of polysaccharide O-acetylation

A family 11 xylanase from the pathogen Botrytis cinerea is inhibited by plant endoxylanase inhibitors XIP-I and TAXI-I

The phytopathogen fungus Botrytis cinerea produces various glycosidases which are secreted during plant infection. In this study, the XynBc1 cDNA that encodes a xylanase from family 11 glycoside hydrolase from B. cinerea was identified by homology-based analysis, cloned by reverse transcription RT-PCR, fully sequenced, and heterologously expressed in Pichia pastoris X-33. The purified recombinant protein obtained by chelating-affinity chromatography demonstrated high catalytic activity (180 ± 23 U/mg) and efficiently degraded low viscosity xylan [Km = 10±3 g L1, Vmax = 0.50 ± 0.04 lmol xylose min1, and kcat = 136 ± 11.5 s1 at pH 4.5 and 25 C]. XynBc1 was further tested for its ability to interact with wheat XIP and TAXI type xylanase inhibitors which have been implicated in plant defence. The xylanase activity of XynBc1 produced in P. pastoris was strongly inhibited by both XIP-I and TAXI-I in a competitive manner, with a Ki of 2.1 ± 0.1 and 6.0 ± 0.2 nM, respectively, whereas no inhibition was detected with TAXI-II. We also showed that XynBc1 mRNAs accumulated during early stages of plant tissue infection