L'hybridation in situ, un outil moléculaire au service de la biologie des plantes

Conférencière invité

"Démasquer le messager" : l'hybridation in situ comme outil moléculaire au service de la biologie des plantes

Conférencière invitée"Rouge ou Verte, l'histocytologie se structure à Montpellier avec la création du " Réseau d'Histologie Expérimentale de Montpellier" (RHEM) et de la plate-forme d'Histocytologie et d'Imagerie Cellulaire Végétale (PHIV) . L'histologie est une science ancienne en plein renouveau. Elle sait combiner recettes du passé et nouvelles technologies, de marquage, de préparation des échantillons, d'observation par microscopie. L'avènement de l'image numérique conduit au développement de la cytomorphométrie qui génère des données quantitatives à partir des préparations histologiques. Aujourd'hui plus qu'hier, l'étude de la structure et de l'organisation spatiale des tissus est l'une des clés des sciences du vivant pour la compréhension des relations structure-fonction. L'objectif de cette journée co-organisée par le RHEM et PHIV est de favoriser les échanges entre histocytologie animale et végétale autour des approches méthodologiques et conceptuelles et de réfléchir ensemble sur les apports de l'histocytologie dans les sciences du vivant.

Secondary metabolite localization by autofluorescence in living plant cells.

International audienceAutofluorescent molecules are abundant in plant cells and spectral images offer means for analyzing their spectra, yielding information on their accumulation and function. Based on their fluorescence characteristics, an imaging approach using multiphoton microscopy was designed to assess localization of the endogenous fluorophores in living plant cells. This method, which requires no previous treatment, provides an effective experimental tool for discriminating between multiple naturally-occurring fluorophores in living-tissues. Combined with advanced Linear Unmixing, the spectral analysis extends the possibilities and enables the simultaneous detection of fluorescent molecules reliably separating overlapping emission spectra. However, as with any technology, the possibility for artifactual results does exist. This methodological article presents an overview of the applications of tissular and intra-cellular localization of these intrinsic fluorophores in leaves and fruits (here for coffee and vanilla). This method will provide new opportunities for studying cellular environments and the behavior of endogenous fluorophores in the intracellular environment

Identification of the Endodermal Vacuole as the Iron Storage Compartment in the Arabidopsis Embryo1

Deciphering how cellular iron (Fe) pools are formed, where they are localized, and which ones are remobilized represents an important challenge to better understand Fe homeostasis. The recent development of imaging techniques, adapted to plants, has helped gain insight into these events. We have analyzed the localization of Fe during embryo development in Arabidopsis (Arabidopsis thaliana) with an improved histochemical staining based on Perls coloration intensified by a second reaction with diaminobenzidine and hydrogen peroxide. The procedure, quick to set up and specific for Fe, was applied directly on histological sections, which dramatically increased its subcellular resolution. We have thus unambiguously shown that in dry seeds Fe is primarily stored in the endodermis cell layer, within the vacuoles, from which it is remobilized during germination. In the vit1-1 mutant, in which the Fe pattern is disturbed, Fe is stored in vacuoles of cortex cells of the hypocotyl/radicle axis and in a single subepidermal cell layer in the cotyledons. During the early stages of embryo development, Fe is evenly distributed in the cells of both wild-type and vit1-1 mutants. Fe eventually accumulates in endodermal cells as the vascular system develops, a process that is impaired in vit1-1. Our results have uncovered a new role for the endodermis in Fe storage in the embryo and have established that the Perls/diaminobenzidine staining is a method of choice to detect Fe in plant tissues and cells

Straightforward histochemical staining of Fe by the adaptation of an old-school technique: Identification of the endodermal vacuole as the site of Fe storage in Arabidopsis embryos

Iron (Fe) is an essential metal ion, required for basic cellular processes such as respiration, photosynthesis and cell division. Therefore, Fe has to be stored and distributed to several organelles to fulfill its roles. The molecular basis of Fe distribution is poorly understood. In this context, elemental imaging approaches are becoming essential for a better understanding of metal homeostasis in plants. Recently, several genes have been involved in Fe storage (VIT1) and remobilization (NRAMP3 and NRAMP4) in the seed of Arabidopsis, mostly with the help of sophisticated imaging techniques. We have adapted an histochemical procedure to detect Fe in plant tissues, based on Perls staining coupled to diaminobenzidine (DAB) intensification. The Perls/DAB technique, quick and inexpensive, was shown to be specific for Fe and highly sensitive. We have applied this procedure to Arabidopsis embryos and shown that Fe is stored in the vacuoles of a specific cell layer surrounding the pro-vascular system, the endodermis. Our results have revealed a new role for the endodermis in Fe storage in the embryo and established the Perls/DAB technique as a powerful tool to detect Fe in plant tissues and cells

Molecular and Physiological Responses to Water Deficit in Drought-Tolerant and Drought-Sensitive Lines of Sunflower : Accumulation of Dehydrin Transcripts Correlates with Tolerance

To investigate correlations between phenotypic adaptation to water limitation and drought-induced gene expression, we have studied a model system consisting of a drought-tolerant line (R1) and a drought-sensitive line (S1) of sunflowers (Helianthus annuus L.) subjected to progressive drought. R1 tolerance is characterized by the maintenance of shoot cellular turgor. Drought-induced genes (HaElip1, HaDhn1, and HaDhn2) were previously identified in the tolerant line. The accumulation of the corresponding transcripts was compared as a function of soil and leaf water status in R1 and S1 plants during progressive drought. In leaves of R1 plants the accumulation of HaDhn1 and HaDhn2 transcripts, but not HaElip1 transcripts, was correlated with the drought-adaptive response. Drought-induced abscisic acid (ABA) concentration was not associated with the varietal difference in drought tolerance. Stomata of both lines displayed similar sensitivity to ABA. ABA-induced accumulation of HaDhn2 transcripts was higher in the tolerant than in the sensitive genotype. HaDhn1 transcripts were similarly accumulated in the tolerant and in the sensitive plants in response to ABA, suggesting that additional factors involved in drought regulation of HaDhn1 expression might exist in tolerant plants

The Arabidopsis Yellow Stripe LIKE4 and 6 transporters control iron release from the chloroplast.

International audienceIn most plant cell types, the chloroplast represents the largest sink for iron, which is both essential for chloroplast metabolism and prone to cause oxidative damage. Here, we show that to buffer the potentially harmful effects of iron, besides ferritins for storage, the chloroplast is equipped with specific iron transporters that respond to iron toxicity by removing iron from the chloroplast. We describe two transporters of the YELLOW STRIPE1-LIKE family from Arabidopsis thaliana, YSL4 and YSL6, which are likely to fulfill this function. Knocking out both YSL4 and YSL6 greatly reduces the plant's ability to cope with excess iron. Biochemical and immunolocalization analyses showed that YSL6 resides in the chloroplast envelope. Elemental analysis and histochemical staining indicate that iron is trapped in the chloroplasts of the ysl4 ysl6 double mutants, which also accumulate ferritins. Also, vacuolar iron remobilization and NRAMP3/4 expression are inhibited. Furthermore, ubiquitous expression of YSL4 or YSL6 dramatically reduces plant tolerance to iron deficiency and decreases chloroplastic iron content. These data demonstrate a fundamental role for YSL4 and YSL6 in managing chloroplastic iron. YSL4 and YSL6 expression patterns support their physiological role in detoxifying iron during plastid dedifferentiation occurring in embryogenesis and senescence

Phenol homeostasis is ensured in vanilla fruit by storage under solid form in a new chloroplast-derived organelle, the phenyloplast.

International audienceA multiple cell imaging approach combining immunofluorescence by confocal microscopy, fluorescence spectral analysis by multiphotonic microscopy, and transmission electron microscopy identified the site of accumulation of 4-O-(3-methoxybenzaldehyde) β-d-glucoside, a phenol glucoside massively stockpiled by vanilla fruit. The glucoside is sufficiently abundant to be detected by spectral analysis of its autofluorescence. The convergent results obtained by these different techniques demonstrated that the phenol glucoside accumulates in the inner volume of redifferentiating chloroplasts as solid amorphous deposits, thus ensuring phenylglucoside cell homeostasis. Redifferentiation starts with the generation of loculi between thylakoid membranes which are progressively filled with the glucoside until a fully matured organelle is obtained. This peculiar mode of storage of a phenolic secondary metabolite is suspected to occur in other plants and its generalization in the Plantae could be considered. This new chloroplast-derived organelle is referred to as a 'phenyloplast'

New insights into Fe localization in plant tissues.

International audienceDeciphering cellular iron (Fe) homeostasis requires having access to both quantitative and qualitative information on the subcellular pools of Fe in tissues and their dynamics within the cells. We have taken advantage of the Perls/DAB Fe staining procedure to perform a systematic analysis of Fe distribution in roots, leaves and reproductive organs of the model plant Arabidopsis thaliana, using wild-type and mutant genotypes affected in iron transport and storage. Roots of soil-grown plants accumulate iron in the apoplast of the central cylinder, a pattern that is strongly intensified when the citrate effluxer FRD3 is not functional, thus stressing the importance of citrate in the apoplastic movement of Fe. In leaves, Fe level is low and only detected in and around vascular tissues. In contrast, Fe staining in leaves of iron-treated plants extends in the surrounding mesophyll cells where Fe deposits, likely corresponding to Fe-ferritin complexes, accumulate in the chloroplasts. The loss of ferritins in the fer1,3,4 triple mutant provoked a massive accumulation of Fe in the apoplastic space, suggesting that in the absence of iron buffering in the chloroplast, cells activate iron efflux and/or repress iron influx to limit the amount of iron in the cell. In flowers, Perls/DAB staining has revealed a major sink for Fe in the anthers. In particular, developing pollen grains accumulate detectable amounts of Fe in small-size intracellular bodies that aggregate around the vegetative nucleus at the binuclear stage and that were identified as amyloplasts. In conclusion, using the Perls/DAB procedure combined to selected mutant genotypes, this study has established a reliable atlas of Fe distribution in the main Arabidopsis organs, proving and refining long-assumed intracellular locations and uncovering new ones. This "iron map" of Arabidopsis will serve as a basis for future studies of possible actors of iron movement in plant tissues and cell compartments