Chloroplast to chromoplast transition in tomato fruit: spectral confocalmicroscopy analyses of carotenoids and chlorophylls in isolated plastids and time-lapse recording on intact live tissue

During evolution, chromoplasts have emerged as plastid structures which accumulate pigments to facilitate flower pollination and seed dispersal of fleshy fruit. There is good evidence that chromoplasts derive from chloroplasts (Pyke, 2007), even if nobody has ever recorded this transition. Structural changes occurring during chloroplast to chromoplast transition have been described in fleshy fruit by electron microscopy primarily in tomato (Rosso, 1968; Harris and Spurr, 1969) and in bell pepper (Spurr and Harris, 1968). During the differentiation process controlled breakdown of chlorophyll and disruption of the thylakoid membrane occurred, concomitant with an increase in the aggregation of carotenoids. Different carotenoid-accumulating bodies have been described, including plastoglobules, crystalline and microfibrillar structures, and internal membranous structures

Tomato EF-Tsmt, a functional mitochondrial translation elongation factor from higher plants

Ethylene-induced ripening in tomato (Lycopersicon esculentum) resulted in the accumulation of a transcript designated LeEF-Tsmt that encodes a protein with significant homology to bacterial Ts translational elongation factor (EF-Ts). Transient expression in tobacco and sunflower protoplasts of full-length and truncated LeEF-Tsmt- GFP fusion constructs and confocal microscopy observations clearly demonstrated the targeting of LeEF-Tsmt to mitochondria and not to chloroplasts and the requirement for a signal peptide for the proper sorting of the protein. Escherichia coli recombinant LeEF-Tsmt co-eluted from Ni-NTA resins with a protein corresponding to the molecular weight of the elongation factor EF-Tu of E. coli, indicating an interaction with bacterial EF-Tu. Increasing the GDP concentration in the extraction buffer reduced the amount of EF-Tu in the purified LeEF-Tsmt fraction. The purified LeEF-Tsmt stimulated the poly(U)-directed polymerization of phenylalanine 10-fold in the presence of EF-Tu. Furthermore, LeEF-Tsmt was capable of catalysing the nucleotide exchange reaction with E. coli EF-Tu. Altogether, these data demonstrate that LeEF-Tsmt encodes a functional mitochondrial EF-Ts. LeEFTsmt represents the first mitochondrial elongation factor to be isolated and functionally characterized in higher plants

Characterisation of sunflower root colonisation by Phoma macdonaldii

Phoma macdonaldii, the causal agent of black stem disease of sunflower (Helianthus annuus), also attacks roots and collars of the plants, resulting in early death. Totally resistant lines do not exist for infection of the aerial parts, but tolerant lines have been characterised. This paper presents a study on colonisation of a partially resistant and a susceptible sunflower line by P. macdonaldii. The fungus was transformed with a constitutively expressed reporter gene encoding the jellyfish green fluorescent protein via Agrobacterium tumefaciens, and colonisation of sunflower roots by this transformed strain was studied by various microscopy techniques including confocal and scanning electron microscopy. The results show that penetration of the fungus into the root occurred through natural fissures or through the epidermis and was similar in both lines. In contrast, the colonisation rate of the stele was reduced in the partially resistant line, and the morphology of the fungal hyphae was also affected. The effect on hyphal morphology was strongest in the stele, indicating a localised production of defence compounds in this line

Maize cell wall degradability, from whole plant to tissue level: different scales of complexity

Today, maize stover can be considered as a model for investigating secondary cell wall formation in grasses with major applications in cattle feeding (forage maize) and green energy production (bioethanol, biogas, etc). Up until now, cell wall formation and cell wall degradability have been considered at the whole plant scale. However, a detailed examination of leaves and internodes has underlined a large diversity of lignified cell types (xylem vessels, parenchyma, sub-epidermal and perivascular sclerenchyma) and significant variations in the organization and / or the composition of these different cell types. In this review, we highlighted several aspects of this complexity and their consequences on valorization processes both in agriculture or industries

Pectin Demethylesterification Generates Platforms that Anchor Peroxidases to Remodel Plant Cell Wall Domains

International audiencePlant cell walls are made of polysaccharidic-proteinaceous complex matrices. Molecular interactions governing their organization remain understudied. We take advantage of the highly dynamic cell walls of Arabidopsis seed mucilage secretory cells to propose a hierarchical multi-molecular interaction model within a cell wall domain. We show that the PECTINMETHYLESTERASE INHIBITOR6 activity creates a partially demethylesterified pectin pattern acting as a platform allowing positioning of PEROXIDASE36 in a remote primary cell wall domain during early development. This allows triggering the loosening of this domain during later development, in turn leading to proper physiological function upon mature seed imbibition and germination. We anticipate that this pioneer example of molecular scaffold within a cell wall domain is more widespread through other combinations of the individual molecular players all belonging to large multigenic families. These results highlight the role of cell wall polysaccharide-protein interactions in the organization of cell wall domains

EgMYB2, a new transcriptional activator from Eucalyptus xylem, regulates secondary cell wall formation and lignin biosynthesis

International audienceEgMYB2, a member of a new subgroup of the R2R3 MYB family of transcription factors, was cloned from a library consisting of RNA from differentiating Eucalyptus xylem. EgMYB2 maps to a unique locus on the Eucalyptus grandis linkage map and co-localizes with a quantitative trait locus (QTL) for lignin content. Recombinant EgMYB2 protein was able to bind specifically the cis-regulatory regions of the promoters of two lignin biosynthetic genes, cinnamoyl-coenzyme A reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), which contain MYB consensus binding sites. EgMYB2 was also able to regulate their transcription in both transient and stable expression assays. Transgenic tobacco plants over-expressing EgMYB2 displayed phenotypic changes relative to wild-type plants, among which were a dramatic increase in secondary cell wall thickness, and an alteration of the lignin profiles. Transcript abundance of genes encoding enzymes specific to lignin biosynthesis was increased to varying extents according to the position of individual genes in the pathway,whereas core phenylpropanoid geneswere not significantly affected. Together these results suggest a role for EgMYB2 in the co-ordinated control of genes belonging to the monolignol-specific pathway, and therefore in the biosynthesis of lignin and the regulation of secondary cell wall formation

Gene profiling in white blood cells predicts infliximab responsiveness in rheumatoid arthritis

As indicators of responsiveness to a tumour necrosis factor (TNF)α blocking agent (infliximab) are lacking in rheumatoid arthritis, we have used gene profiling in peripheral blood mononuclear cells to predict a good versus poor response to infliximab. Thirty three patients with very active disease (Disease Activity Score 28 >5.1) that resisted weekly methotrexate therapy were given infliximab at baseline, weeks 2 and 6, and every 8th week thereafter. The patients were categorized as responders if a change of Disease Activity Score 28 = 1.2 was obtained at 3 months. Mononuclear cell RNAs were collected at baseline and at three months from responders and non-responders. The baseline RNAs were hybridised to a microarray of 10,000 non-redundant human cDNAs. In 6 responders and 7 non-responders, 41 mRNAs identified by microarray analysis were expressed as a function of the response to treatment and an unsupervised hierarchical clustering perfectly separated these responders from non-responders. The informativeness of 20 of these 41 transcripts, as measured by qRT-PCR, was re-assessed in 20 other patients. The combined levels of these 20 transcripts properly classified 16 out of 20 patients in a leave-one-out procedure, with a sensitivity of 90% and a specificity of 70%, whereas a set of only 8 transcripts properly classified 18/20 patients. Trends for changes in various transcript levels at three months tightly correlated with treatment responsiveness and a down-regulation of specific transcript levels was observed in non-responders only. Our gene profiling obtained by a non-invasive procedure should now be used to predict the likely responders to an infliximab/methotrexate combination

Molecular and biochemical characterization of LeCRK1, a ripening-associated tomato CDPK-related kinase

A cDNA clone (LeCRK1), encoding a novel isoform of calcium-dependent protein kinase (CDPK), was isolated by screening a tomato (Lycopersicon esculentum) cDNA library. The protein derived from the full-length sequence indicated that it belongs to the family of CDPK-related kinases (CRKs) and the predicted amino acid sequence shows a modular organization of the protein consisting of different characteristic domains. The kinase domain of LeCRK1 shares a high degree of similarity with the catalytic domain of CDPKs. In contrast to canonical members of the family, LeCRK1 has a degenerate sequence in the C-terminal calmodulinlike domain. LeCRK1 protein was shown to be a functional kinase, but, consistent with the lack of calciumbinding activity, its autophosphorylation activity did not require calcium. LeCRK1 harbours an amphiphilic amino acid region revealed to be a functional calmodulinbinding site by in vitro assay. A putative myristoylation/ palmitoylation sequence has been identified at the N-terminus. Expressing an LeCRK1::GFP fusion protein in the protoplast resulted in its targeting to the plasma membrane. Site-directed mutagenesis of critical amino acids of the myristoylation/palmitoylation consensus sites led to the accumulation of the mutated protein in the cytoplasm, suggesting that the native protein is anchored to the plasma membrane by acylated residues. Expression studies revealed significant accumulation of LeCRK1 transcripts during fruit ripening, although transcripts were also detected in stem, leaf, and flower. LeCRK1 mRNA level in leaves was slightly induced by ethylene and salicylic acid, and upon mechanical wounding and cold treatment. It is noteworthy that LeCRK1 mRNAs were undetectable in different tomato-ripening natural mutants such as NR, Rin, and Nor, suggesting a role in the ripening process

Combined approaches provide an anatomical and transcriptomic fingerprint of maize cell wall digestibility

Understanding cell wall biosynthesis and degradation in grasses has become a major aim in plant biology. Although independent previous reports have focused on specific features that dictate cell wall digestibility, cytological, biochemical, and gene regulation parameters have never been integrated within the same study. Herein, we applied a combination of state-of-the-art technologies and different scales of observation on two maize lines that are characterized by highly contrasted forage digestibility. Comparative image analysis of internode sections allow to get an anatomical fingerprint associated with high digestibility: a thin peripheral rind of lignified parenchyma, small numerous vascular bundles, and low proportion of PeriVascular Sclerenchyma (PVS). This cell type patterning led to enhanced digestibility when internode sections were treated with Celluclast, a commercially cell wall degrading enzyme. At a lower scale of observation, Laser Capture Microdissection (LCM) followed by thioacidolysis of PVS revealed a higher proportion of Syringyl (S) unit lignins in the low digestible line while the high digestible line was p-Hydroxyphenyl (H)-rich. Moreover, cytological observation of internodes of the two lines point out that this difference in composition is associated with a delayed lignification of PVS. At the same time, comparative transcriptomics on internodes indicated differential expression of several genes encoding enzymes along the phenylpropanoid pathway and known cell wall-associated Transcription Factors (TFs). Together, these results give an integrative view of different factors which could aim in designing a maize silage ideotype and provide a novel set of potential regulatory genes controlling lignification in maize