RESUMO
Plant myxodiasporous species have the ability to release a polysaccharidic mucilage upon imbibition of the seed (myxospermy) or the fruit (myxocarpy). This is a widespread capacity in angiosperms providing multiple ecological functions including higher germination efficiency under environmental stresses. It is unclear whether myxodiaspory has one or multiple evolutionary origins and why it was supposedly lost in several species. Here, we summarize recent advances on three main aspects of myxodiaspory. (a) It represents a combination of highly diverse traits at different levels of observation, ranging from the dual tissular origin of mucilage secretory cells to diverse mucilage polysaccharidic composition and ultrastructural organization. (b) An asymmetrical selection pressure is exerted on myxospermy-related genes that were first identified in Arabidopsis thaliana. The A. thaliana and the flax intra-species mucilage variants show that myxospermy is a fast-evolving trait due to high polymorphism in a few genes directly acting on mucilage establishment. In A. thaliana, these actors are downstream of a master regulatory complex and an original phylogenetic overview provided here illustrates that this complex has sequentially evolved after the common ancestor of seed plants and was fully established in the common ancestor of the rosid clade. (c) Newly identified myxodiaspory ecological functions indicate new perspectives such as soil microorganism control and plant establishment support.
Assuntos
Evolução Biológica , Mucilagem Vegetal/fisiologia , Sementes/fisiologia , Adaptação Fisiológica/fisiologia , Ecologia , Meio Ambiente , Mucilagem Vegetal/metabolismo , Sementes/metabolismo , Estresse Fisiológico/fisiologiaRESUMO
Exogenous low pH stress causes cell death in root cells, limiting root development, and agricultural production. Different lines of evidence suggested a relationship with cell wall (CW) remodeling players. We investigated whether class III peroxidase (CIII Prx) total activity, CIII Prx candidate gene expression, and reactive oxygen species (ROS) could modify CW structure during low pH-induced cell death in Arabidopsis thaliana roots. Wild-type roots displayed a good spatio-temporal correlation between the low pH-induced cell death and total CIII Prx activity in the early elongation (EZs), transition (TZs), and meristematic (MZs) zones. In situ mRNA hybridization showed that AtPrx62 transcripts accumulated only in roots treated at pH 4.6 in the same zones where cell death was induced. Furthermore, roots of the atprx62-1 knockout mutant showed decreased cell mortality under low pH compared to wild-type roots. Among the ROS, there was a drastic decrease in O2·- levels in the MZs of wild-type and atprx62-1 roots upon low pH stress. Together, our data demonstrate that AtPrx62 expression is induced by low pH and that the produced protein could positively regulate cell death. Whether the decrease in O2·- level is related to cell death induced upon low pH treatment remains to be elucidated.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Morte Celular/genética , Raízes de Plantas/genética , Arabidopsis/crescimento & desenvolvimento , Parede Celular/genética , Regulação da Expressão Gênica de Plantas/genética , Concentração de Íons de Hidrogênio , Meristema/genética , Meristema/crescimento & desenvolvimento , Oxirredução/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismoRESUMO
The iridoids comprise a large family of distinctive bicyclic monoterpenes that possess a wide range of pharmacological activities, including anticancer, anti-inflammatory, antifungal and antibacterial activities. Additionally, certain iridoids are used as sex pheromones in agriculturally important species of aphids, a fact that has underpinned innovative and integrated pest management strategies. To harness the biotechnological potential of this natural product class, the enzymes involved in the biosynthetic pathway must be elucidated. Here we report the discovery of iridoid synthase, a plant-derived enzyme that generates the iridoid ring scaffold, as evidenced by biochemical assays, gene silencing, co-expression analysis and localization studies. In contrast to all known monoterpene cyclases, which use geranyl diphosphate as substrate and invoke a cationic intermediate, iridoid synthase uses the linear monoterpene 10-oxogeranial as substrate and probably couples an initial NAD(P)H-dependent reduction step with a subsequent cyclization step via a Diels-Alder cycloaddition or a Michael addition. Our results illustrate how a short-chain reductase was recruited as cyclase for the production of iridoids in medicinal plants. Furthermore, we highlight the prospects of using unrelated reductases to generate artificial cyclic scaffolds. Beyond the recognition of an alternative biochemical mechanism for the biosynthesis of cyclic terpenes, we anticipate that our work will enable the large-scale heterologous production of iridoids in plants and microorganisms for agricultural and pharmaceutical applications.
Assuntos
Biocatálise , Catharanthus/enzimologia , Iridoides/química , Iridoides/metabolismo , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/metabolismo , Produtos Biológicos/química , Produtos Biológicos/metabolismo , Catharanthus/genética , Catharanthus/metabolismo , Ciclização , Reação de Cicloadição , Dados de Sequência Molecular , Monoterpenos/metabolismo , NADP/metabolismo , Oxirredutases/metabolismo , Extratos Vegetais/química , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Medicinais/enzimologia , Plantas Medicinais/genética , Plantas Medicinais/metabolismo , Especificidade por SubstratoRESUMO
Several physiological functions have been attributed to class III peroxidases (PRXs) in plants, but the in planta role of most members of this family still remains undetermined. Here, we report the first functional characterization of PRX17 (At2g22420), one of the 73 members of this family in Arabidopsis thaliana. Localization of PRX17 was examined by transient expression in Nicotiana benthamiana. Loss- and gain-of-function mutants in A. thaliana were studied. Regulation at the gene and protein levels was analyzed using ß-glucuronidase (GUS) activity, quantitative reverse transcriptase (qRT)-PCR, zymography, and chromatin immunoprecipitation. Phenotypes were characterized including lignin and xylan contents. PRX17 was expressed in various tissues, including vascular tissues, and PRX17 was localized to the cell wall. In prx17, the lignin content was reduced in the stem and siliques and bolting was delayed, while the opposite phenotype was observed in 35S:PRX17 plants, together with a significant increase of lignin and xylan immunofluorescence signal. Finally, we demonstrated that the transcription factor AGAMOUS-LIKE15 (AGL15) binds to the PRX17 promoter and regulates PRX17 expression level. This converging set of structural, transcriptomic and physiological data suggests that PRX17, under the control of AGL15, contributes to developmental programs by playing an essential role in regulating age-dependent lignified tissue formation, including changes in cell wall properties.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Lignina/metabolismo , Proteínas de Domínio MADS/metabolismo , Peroxidase/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , DNA Bacteriano/genética , Flores/fisiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Domínio MADS/genética , Mutação/genética , Peroxidases , Filogenia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismoRESUMO
Expansion of the biosynthesis of plant specialized metabolites notably results from the massive recruitment of cytochrome P450s that catalyze multiple types of conversion of biosynthetic intermediates. For catalysis, P450s require a two-electron transfer catalyzed by shared cytochrome P450 oxidoreductases (CPRs), making these auxiliary proteins an essential component of specialized metabolism. CPR isoforms usually group into two distinct classes with different proposed roles, namely involvement in primary and basal specialized metabolisms for class I and inducible specialized metabolism for class II. By studying the role of CPRs in the biosynthesis of monoterpene indole alkaloids, we provide compelling evidence of an operational specialization of CPR isoforms in Catharanthus roseus (Madagascar periwinkle). Global analyses of gene expression correlation combined with transcript localization in specific leaf tissues and gene-silencing experiments of both classes of CPR all point to the strict requirement of class II CPRs for monoterpene indole alkaloid biosynthesis with a minimal or null role of class I. Direct assays of interaction and reduction of P450s in vitro, however, showed that both classes of CPR performed equally well. Such high specialization of class II CPRs in planta highlights the evolutionary strategy that ensures an efficient reduction of P450s in specialized metabolism.
Assuntos
Alcaloides/biossíntese , Vias Biossintéticas , Catharanthus/enzimologia , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Biocatálise , Vias Biossintéticas/genética , Catharanthus/genética , Cotilédone/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas , Alcaloides Indólicos/metabolismo , NADPH-Ferri-Hemoproteína Redutase/química , NADPH-Ferri-Hemoproteína Redutase/genética , Folhas de Planta/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Frações Subcelulares/enzimologiaRESUMO
Hydroxylation of tabersonine at the C-16 position, catalyzed by tabersonine 16-hydroxylase (T16H), initiates the synthesis of vindoline that constitutes the main alkaloid accumulated in leaves of Catharanthus roseus. Over the last decade, this reaction has been associated with CYP71D12 cloned from undifferentiated C. roseus cells. In this study, we isolated a second cytochrome P450 (CYP71D351) displaying T16H activity. Biochemical characterization demonstrated that CYP71D12 and CYP71D351 both exhibit high affinity for tabersonine and narrow substrate specificity, making of T16H, to our knowledge, the first alkaloid biosynthetic enzyme displaying two isoforms encoded by distinct genes characterized to date in C. roseus. However, both genes dramatically diverge in transcript distribution in planta. While CYP71D12 (T16H1) expression is restricted to flowers and undifferentiated cells, the CYP71D351 (T16H2) expression profile is similar to the other vindoline biosynthetic genes reaching a maximum in young leaves. Moreover, transcript localization by carborundum abrasion and RNA in situ hybridization demonstrated that CYP71D351 messenger RNAs are specifically located to leaf epidermis, which also hosts the next step of vindoline biosynthesis. Comparison of high- and low-vindoline-accumulating C. roseus cultivars also highlights the direct correlation between CYP71D351 transcript and vindoline levels. In addition, CYP71D351 down-regulation mediated by virus-induced gene silencing reduces vindoline accumulation in leaves and redirects the biosynthetic flux toward the production of unmodified alkaloids at the C-16 position. All these data demonstrate that tabersonine 16-hydroxylation is orchestrated in an organ-dependent manner by two genes including CYP71D351, which encodes the specific T16H isoform acting in the foliar vindoline biosynthesis.
Assuntos
Catharanthus/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Especificidade de Órgãos , Proteínas de Plantas/metabolismo , Vimblastina/análogos & derivados , Biocatálise , Vias Biossintéticas/genética , Catharanthus/citologia , Catharanthus/genética , Sistema Enzimático do Citocromo P-450/genética , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Retículo Endoplasmático/enzimologia , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Genes de Plantas/genética , Hidroxilação , Alcaloides Indólicos/química , Alcaloides Indólicos/metabolismo , Cinética , Metaboloma/genética , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Epiderme Vegetal/citologia , Epiderme Vegetal/enzimologia , Epiderme Vegetal/genética , Proteínas de Plantas/genética , Quinolinas/química , Quinolinas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato , Vimblastina/biossíntese , Vimblastina/químicaRESUMO
The myxospermous species Arabidopsis thaliana extrudes a polysaccharidic mucilage from the seed coat epidermis during imbibition. The whole seed mucilage can be divided into a seed-adherent layer and a fully soluble layer, both layers presenting natural genetic variations. The adherent mucilage is variable in size and composition, while the soluble mucilage is variable in composition and physical properties. Studies reporting both the genetic architecture and the putative selective agents acting on this natural genetic variation are scarce. In this study, we set up a Genome Wide Association study (GWAS) based on 424 natural accessions collected from 166 natural populations of A. thaliana located south-west of France and previously characterized for a very important number of abiotic and biotic factors. We identified an extensive genetic variation for both mucilage layers. The adherent mucilage was mainly related to precipitation and temperature whereas the non-adherent mucilage was unrelated to any environmental factors. By combining a hierarchical Bayesian model with a local score approach, we identified 55 and 28 candidate genes, corresponding to 26 and 10 QTLs for the adherent and non-adherent mucilages, respectively. Putative or characterized function and expression data available in the literature were used to filter the candidate genes. Only one gene among our set of candidate genes was already described as a seed mucilage actor, leaving a large set of new candidates putatively implicated inseed mucilage synthesis or release. The present study lay out foundation to understand the influence of regional ecological factors acting on seed mucilage in A. thaliana.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Mucilagem Vegetal , Arabidopsis/genética , Arabidopsis/metabolismo , Estudo de Associação Genômica Ampla , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Teorema de Bayes , Mucilagem Vegetal/genética , Mucilagem Vegetal/metabolismo , Mutação , Polissacarídeos/metabolismo , Sementes/genética , Sementes/metabolismoRESUMO
Plant cell walls constitute complex polysaccharidic/proteinaceous networks whose biosynthesis and dynamics implicate several cell compartments. The synthesis and remodeling of homogalacturonan pectins involve Golgi-localized methylation/acetylation and subsequent cell wall-localized demethylation/deacetylation. So far, TRICHOME BIREFRINGENCE-LIKE (TBL) family members have been described as Golgi-localized acetyltransferases targeting diverse hemicelluloses or pectins. Using seed mucilage secretory cells (MSCs) from Arabidopsis thaliana, we demonstrate the atypical localization of TBL38 restricted to a cell wall microdomain. A tbl38 mutant displays an intriguing homogalacturonan immunological phenotype in this cell wall microdomain and in an MSC surface-enriched abrasion powder. Mass spectrometry oligosaccharide profiling of this fraction reveals an increased homogalacturonan acetylation phenotype. Finally, TBL38 displays pectin acetylesterase activity in vitro. These results indicate that TBL38 is an atypical cell wall-localized TBL that displays a homogalacturonan acetylesterase activity rather than a Golgi-localized acetyltransferase activity as observed in previously studied TBLs. TBL38 function during seed development is discussed.
RESUMO
Plant cell wall researchers were asked their view on what the major unanswered questions are in their field. This article summarises the feedback that was received from them in five questions. In this issue you can find equivalent syntheses for researchers working on bacterial, unicellular parasite and fungal systems.
RESUMO
Polysaccharides make up about 75% of plant cell walls and can be broken down to produce sugar substrates (saccharification) from which a whole range of products can be obtained, including bioethanol. Cell walls also contain 5-10% of proteins, which could be used to tailor them for agroindustrial uses. Here we present cell wall proteomics data of Brachypodium distachyon, a model plant for temperate grasses. Leaves and culms were analyzed during active growth and at mature stage. Altogether, 559 proteins were identified by LC-MS/MS and bioinformatics, among which 314 have predicted signal peptides. Sixty-three proteins were shared by two organs at two developmental stages where they could play housekeeping functions. Differences were observed between organs and stages of development, especially at the level of glycoside hydrolases and oxidoreductases. Differences were also found between the known cell wall proteomes of B. distachyon, Oryza sativa, and the Arabidopsis thaliana dicot. Three glycoside hydrolases could be immunolocalized in cell walls using polyclonal antibodies against proteotypic peptides. Organ-specific expression consistent with proteomics results could be observed as well as cell-specific localization. Moreover, the high number of proteins of unknown function in B. distachyon cell wall proteomes opens new fields of research for monocot cell walls.
Assuntos
Biocombustíveis , Brachypodium/química , Parede Celular/química , Proteínas de Plantas/análise , Proteoma/análise , Brachypodium/citologia , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/química , Imuno-Histoquímica , Folhas de Planta/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Caules de Planta/química , Proteoma/química , Proteoma/metabolismoRESUMO
Plant primary cell walls are composite structures surrounding the protoplast and containing pectins, hemicelluloses, and cellulose polysaccharides, as well as proteins. Their composition changed during the evolution of the green lineage from algae to terrestrial plants, i.e., from an aquatic to a terrestrial environment. The constraints of life in terrestrial environments have generated new requirements for the organisms, necessitating adaptations, such as cell wall modifications. We have studied the cell wall polysaccharide composition of thalli of Marchantia polymorpha, a bryophyte belonging to one of the first land plant genera. Using a collection of specific antibodies raised against different cell wall polysaccharide epitopes, we were able to identify in polysaccharide-enriched fractions: pectins, including low-methylesterified homogalacturonans; rhamnogalacturonan I with arabinan side-chains; and hemicelluloses, such as xyloglucans with XXLG and XXXG modules, mannans, including galactomannans, and xylans. We could also show the even distribution of XXLG xyloglucans and galactomannans in the cell walls of thalli by immunocytochemistry. These results are discussed with regard to the cell wall proteome composition and in the context of the evolution of the green lineage. The cell wall polysaccharides of M. polymorpha illustrate the transition from the charophyte ancestors of terrestrial plants containing xyloglucans, xylans and mannans as hemicelluloses, and embryophytes which do not exhibit mannans as major primary cell wall polysaccharides.
Assuntos
Embriófitas , Marchantia , Xilanos/metabolismo , Marchantia/metabolismo , Mananas/metabolismo , Polissacarídeos/metabolismo , Pectinas/metabolismo , Embriófitas/química , Embriófitas/metabolismo , Plantas/metabolismo , Parede Celular/metabolismoRESUMO
Elucidating biosynthetic pathways of plant specialized metabolites is a tricky but essential task for the biotechnological production of plant drugs. In a new report, Li et al. used a single-cell multi-omics approach to provide an integrative view of the architecture and regulation of anticancer alkaloid routes in Madagascar periwinkle.
RESUMO
Immunocytochemistry is a widely used technique to localize antigen within intact tissues. Plant cell walls are complex matrixes of highly decorated polysaccharides and the large number of CBM families displaying specific substrate recognition reflects this complexity. The accessibility of large proteins, such as antibodies, to their cell wall epitopes may be sometimes difficult due to steric hindrance problems. Due to their smaller size, CBMs are interesting alternative probes. The aim of this chapter is to describe the use of CBM as probes to explore complex polysaccharide topochemistry in muro and to quantify enzymatic deconstruction.
Assuntos
Parede Celular , Polissacarídeos , Humanos , Imuno-Histoquímica , Parede Celular/metabolismo , Polissacarídeos/metabolismo , Membrana Celular/metabolismoRESUMO
Isopentenyl diphosphate isomerases (IDI) catalyze the interconversion of the two isoprenoid universal C5 units, isopentenyl diphosphate and dimethylally diphosphate, to allow the biosynthesis of the large variety of isoprenoids including both primary and specialized metabolites. This isomerisation is usually performed by two distinct IDI isoforms located either in plastids/peroxisomes or mitochondria/peroxisomes as recently established in Arabidopsis thaliana mainly accumulating primary isoprenoids. By contrast, almost nothing is known in plants accumulating specialized isoprenoids. Here we report the cloning and functional validation of an IDI encoding cDNA (CrIDI1) from Catharanthus roseus that produces high amount of monoterpenoid indole alkaloids. The corresponding gene is expressed in all organs including roots, flowers and young leaves where transcripts have been detected in internal phloem parenchyma and epidermis. The CrIDI1 gene also produces long and short transcripts giving rise to corresponding proteins with and without a N-terminal transit peptide (TP), respectively. Expression of green fluorescent protein fusions revealed that the long isoform is targeted to both plastids and mitochondria with an apparent similar efficiency. Deletion/fusion experiments established that the first 18-residues of the N-terminal TP are solely responsible of the mitochondria targeting while the entire 77-residue long TP is needed for an additional plastid localization. The short isoform is targeted to peroxisomes in agreement with the presence of peroxisome targeting sequence at its C-terminal end. This complex plastid/mitochondria/peroxisomes triple targeting occurring in C. roseus producing specialized isoprenoid secondary metabolites is somehow different from the situation observed in A. thaliana mainly producing housekeeping isoprenoid metabolites.
Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/genética , Catharanthus/enzimologia , Catharanthus/genética , Genes de Plantas , Sequência de Aminoácidos , Sequência de Bases , Isomerases de Ligação Dupla Carbono-Carbono/química , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Clonagem Molecular , DNA de Plantas/genética , Hemiterpenos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Mitocôndrias/enzimologia , Dados de Sequência Molecular , Peroxissomos/enzimologia , Plantas Geneticamente Modificadas , Plastídeos/enzimologia , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Terpenos/metabolismo , Transformação GenéticaRESUMO
Plant cell walls display cellular and subcellular specificities. At the subcellular level, wall regional territories with specific compositions are necessary for macroscopic developmental processes. These regional specificities were named differently throughout the years, and are unified here under the term 'cell-wall microdomains' that define the local composition and organization of wall polymers underlying territories of wall loosening and/or softening or stiffening. We review the occurrence and developmental role of wall microdomains in different cell types. We primarily focus on the contribution of two categories of wall-remodeling molecular actors: fine-tuning of homogalacturonan (HG; pectin) demethylesterification patterns and two classes of oxidoreductases [class III peroxidases (CIII PRXs) and laccases (LACs)], but we also highlight two different molecular scaffolds recently identified for positioning specific CIII PRXs.
Assuntos
Parede Celular , Pectinas , Parede Celular/metabolismo , Pectinas/metabolismo , Peroxidases/metabolismoRESUMO
As a mean to cope with their potential cytotoxicity for the host plant, secondary metabolisms are often sequestered within specific cell types. This spatial organization may reach complex sequential multicellular compartmentation. The most complex example so far characterized is the sequential multicellular biosynthesis of the anticancer monoterpene indole alkaloids in Catharanthus roseus. RNA in situ hybridization has proven a key technological approach to unravel this complex spatial organization. Pioneer work in 1999 discovered the involvement of epidermis and laticifer/idioblasts in the intermediate and late steps of the pathway, respectively. The localization of the early steps of the pathway to the internal phloem-associated parenchyma later came to complete the three-tissular block organization of the pathway. Since then, RNA in situ hybridization was routinely used to map the gene expression profile of most of the nearly 30 genes involved in this pathway. We introduce here a comparison of advantages and drawbacks of in situ hybridization and more popular promoter: GUS strategies. Two main advantages of in situ hybridization are the suitability to any plant species and the direct localization of transcripts rather than the localization of a promoter activity. We provide a step-by-step protocol describing every details allowing to reach a medium throughput including riboprobe synthesis, paraffin-embedded plant tissue array preparation, prehybridization, in situ hybridization, stringent washing and immunodetection of hybridized probes, and imaging steps. This should be helpful for new comers willing to domesticate the technique. This protocol has no species limitation and is particularly adapted to the increasingly studied model, nonmodel species, nonamenable to promoter::GUS transformation, such as C. roseus.
Assuntos
Catharanthus , Parafina , Catharanthus/genética , Catharanthus/metabolismo , Hibridização In Situ , RNA/metabolismo , Metabolismo SecundárioRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0150539.].
RESUMO
The ability to extrude mucilage upon seed imbibition (myxospermy) occurs in several Angiosperm taxonomic groups, but its ancestral nature or evolutionary convergence origin remains misunderstood. We investigated seed mucilage evolution in the Brassicaceae family with comparison to the knowledge accumulated in Arabidopsis thaliana. The myxospermy occurrence was evaluated in 27 Brassicaceae species. Phenotyping included mucilage secretory cell morphology and topochemistry to highlight subtle myxospermy traits. In parallel, computational biology was driven on the one hundred genes constituting the so-called A. thaliana mucilage secretory cell toolbox to confront their sequence conservation to the observed phenotypes. Mucilage secretory cells show high morphology diversity; the three studied Arabidopsis species had a specific extrusion modality compared to the other studied Brassicaceae species. Orthologous genes from the A. thaliana mucilage secretory cell toolbox were mostly found in all studied species without correlation with the occurrence of myxospermy or even more sub-cellular traits. Seed mucilage may be an ancestral feature of the Brassicaceae family. It consists of highly diverse subtle traits, probably underlined by several genes not yet characterized in A. thaliana or by species-specific genes. Therefore, A. thaliana is probably not a sufficient reference for future myxospermy evo-devo studies.
Assuntos
Arabidopsis/química , Evolução Biológica , Brassicaceae/química , Mucilagem Vegetal/química , Proteínas de Plantas/metabolismo , Sementes/química , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Brassicaceae/genética , Brassicaceae/crescimento & desenvolvimento , Parede Celular , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Filogenia , Proteínas de Plantas/genética , Sementes/genética , Sementes/crescimento & desenvolvimento , TranscriptomaRESUMO
Irrespective of their biological origin, most proteins are composed of several elementary domains connected by linkers. These domains are either functionally independent units, or part of larger multidomain structures whose functions are defined by their spatial proximity. Carbohydrate-degrading enzymes provide examples of a range of multidomain structures, in which catalytic protein domains are frequently appended to one or more non-catalytic carbohydrate-binding modules which specifically bind to carbohydrate motifs. While the carbohydrate-binding specificity of these modules is clear, their function is not fully elucidated. Herein, an original approach to tackle the study of carbohydrate-binding modules using the Jo-In biomolecular welding protein pair is presented. To provide a proof of concept, recombinant xylanases appended to two different carbohydrate-binding modules have been created and produced. The data reveal the biochemical properties of four xylanase variants and provide the basis for correlating enzyme activity to structural properties and to the nature of the substrate and the ligand specificity of the appended carbohydrate-binding module. It reveals that specific spatial arrangements favour activity on soluble polymeric substrates and that activity on such substrates does not predict the behaviour of multimodular enzymes on insoluble plant cell wall samples. The results highlight that the Jo-In protein welding system is extremely useful to design multimodular enzyme systems, especially to create rigid conformations that decrease the risk of intermodular interference. Further work on Jo-In will target the introduction of varying degrees of flexibility, providing the means to study this property and the way it may influence multimodular enzyme functions.
Assuntos
Parede Celular , Endo-1,4-beta-Xilanases , Células Vegetais/enzimologia , Engenharia de Proteínas , Carboidratos , Domínio Catalítico , Parede Celular/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Especificidade por SubstratoRESUMO
All-flesh tomato cultivars are devoid of locular gel and exhibit enhanced firmness and improved postharvest storage. Here, we show that SlMBP3 is a master regulator of locular tissue in tomato fruit and that a deletion at the gene locus underpins the All-flesh trait. Intriguingly, All-flesh varieties lack the deleterious phenotypes reported previously for SlMBP3 under-expressing lines and which preclude any potential commercial use. We resolve the causal factor for this phenotypic divergence through the discovery of a natural mutation at the SlAGL11 locus, a close homolog of SlMBP3. Misexpressing SlMBP3 impairs locular gel formation through massive transcriptomic reprogramming at initial phases of fruit development. SlMBP3 influences locule gel formation by controlling cell cycle and cell expansion genes, indicating that important components of fruit softening are determined at early pre-ripening stages. Our findings define potential breeding targets for improved texture in tomato and possibly other fleshy fruits.